Engineered pesticidal proteins and methods of controlling plant pests

ABSTRACT

The invention provides nucleic acids, polypeptides, transgenic plants, compositions and methods for conferring pesticidal activity (e.g., insecticidal activity) to bacteria, plants, plant cells, tissues and seeds. Nucleic acids encoding the insecticidal proteins can be used to transform prokaryotic and eukaryotic organisms, including plants, to express the insecticidal proteins. The recombinant organisms and compositions containing the recombinant organisms or insecticidal proteins can be used to control a pest (e.g., an insect).

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. application Ser. No.16/648,323, filed Mar. 18, 2020, which is a national stage applicationof international application No. PCT/US2018/053687, filed on Oct. 1,2018 and published as WO2019/070554 on Apr. 11, 2019, which is entitledto the benefit of U.S. Provisional Application No. 62/566,692, filed onOct. 2, 2017, all of which are incorporated herein by reference in theirentirety.

STATEMENT REGARDING ELECTRONIC SUBMISSION OF A SEQUENCE LISTING

A Sequence Listing in XML format, submitted under 37 C.F.R. § 1.831(a),entitled “81453-US-DIV1 sequence listing.xml”, 299,008 bytes in size,generated on Jan. 30, 2023 and filed via EFS-Web is provided in lieu ofa paper copy. This Sequence Listing is hereby incorporated by referenceinto the specification for its disclosure.

FIELD OF THE INVENTION

This invention relates to engineered pesticidal proteins and the nucleicacid molecules that encode them, as well as compositions and methods forcontrolling plant pests.

BACKGROUND

Insect pests are a major cause of crop losses. In the US alone, billionsof dollars are lost every year due to infestation by various genera ofinsects. In addition to losses in field crops, insect pests are also aburden to vegetable and fruit growers, to producers of ornamentalflowers, and they are a nuisance to gardeners and homeowners.

Species of corn rootworm are considered to be the most destructive cornpests. In the United States, the three important species are Diabroticavirgifera virgifera, the western corn rootworm (WCRW), D. longicornisbarberi, the northern corn rootworm (NCRW and D. undecimpunctatahowardi, the southern corn rootworm (SCRW). Only western and northerncorn rootworms are considered primary pests of corn in the US Corn Belt.Additionally, an important corn rootworm pest in the Southern US is theMexican corn rootworm, Diabrotica virgifera zeae (MCRW). Corn rootwormlarvae cause the most substantial plant damage by feeding almostexclusively on corn roots. This injury has been shown to increase plantlodging, to reduce grain yield and vegetative yield as well as alter thenutrient content of the grain. Larval feeding also causes indirecteffects on corn by opening avenues through the roots for bacterial andfungal infections which lead to root and stalk rot diseases. Adult cornrootworms are active in cornfields in late summer where they feed onears, silks and pollen, thus interfering with normal pollination.

Corn rootworms are mainly controlled by intensive applications ofchemical pesticides, which are active through inhibition of insectgrowth, prevention of insect feeding or reproduction, or cause death.Good corn rootworm control can thus be reached, but these chemicals cansometimes also affect other, beneficial organisms. Another problemresulting from the wide use of chemical pesticides is the appearance ofresistant insect varieties. Yet another problem is due to the fact thatcorn rootworm larvae feed underground thus making it difficult to applyrescue treatments of insecticides. Therefore, most insecticideapplications are made prophylactically at the time of planting. Thispractice results in a large environmental burden. This has beenpartially alleviated by various farm management practices, but there isan increasing need for alternative pest control mechanisms.

Biological pest control agents, such as Bacillus thuringiensis (Bt)strains expressing pesticidal toxins like delta-endotoxins (also calledcrystal toxins or Cry proteins), have been applied to crop plants withsatisfactory results against insect pests. The delta-endotoxins areproteins held within a crystalline matrix that are known to possessinsecticidal activity when ingested by certain insects. Several nativeCry proteins from Bacillus thuringiensis, or engineered Cry proteins,have been expressed in transgenic crop plants and exploited commerciallyto control certain lepidopteran and coleopteran insect pests. Forexample, starting in 2003, transgenic corn hybrids that control cornrootworm by expressing a Cry3Bb1 (e.g., in corn event MON88017),Cry34Ab1/Cry35Ab1 (e.g., in corn event DAS-59122) or modified Cry3A(mCry3A; e.g., in corn event MIR604) or Cry3Ab (eCry3.1Ab; e.g., in cornevent MIR604) protein have been available commercially in the US.

Although the usage of transgenic plants expressing Cry proteins has beenshown to be extremely effective, insect pests that now have resistanceagainst the Cry proteins expressed in certain transgenic plants areknown. Therefore, there remains a need to identity new and effectivepest control agents that provide an economic benefit to farmers and thatare environmentally acceptable. Particularly needed are proteins thatare toxic to Diabrotica species, a major pest of corn, that have adifferent mode of action than existing insect control products as a wayto mitigate the development of resistance. Furthermore, delivery ofinsect control agents through products that minimize the burden on theenvironment, as through transgenic plants, are desirable.

SUMMARY OF THE INVENTION

The invention provides nucleic acids, polypeptides, compositions andmethods for conferring pesticidal activity (e.g., insecticidal activity)to bacteria, plants, plant cells, tissues and seeds. In particular, theinvention provides novel engineered pesticidal proteins (e.g.,engineered insecticidal proteins), optionally with altered or enhancedpesticidal (e.g., insecticidal) activity and/or processing (i.e.,cleavage) by mammalian digestive proteases as compared with the parentmolecule (i.e., an Axmi205 protein that does not comprise a modificationaccording to the present invention).

In embodiments, the engineered proteins of the invention are toxic toeconomically important insect pests (e.g., by inhibiting the ability ofthe insect pest to survive, grow and/or reproduce), particularly insectpests that infest plants. For example, in embodiments, the insecticidalproteins of the invention can be used to control one or moreeconomically important coleopteran pests including without limitationBean Leaf Beetle (Cerotoma trifurcata), Colorado Potato Beetle(Leptinotarsa decemlineata), Boll Weevil (Anthonomus grandis) and/or acorn rootworm pest (e.g., Diabrotica spp.), for example, Western CornRootworm (WCRW; Diabrotica virgifera virgifera), Northern Corn Rootworm(NCRW; D. barberi), Southern Corn Rootworm (D. undecimpunctata howardi),Mexican Corn Rootworm (MCRW; D. virgifera zeae), and the like. Inembodiments, the insecticidal protein has activity against a WCRW pestthat is resistant to a mCry3A protein (e.g., in corn event MIR604), aeCry3.1Ab protein (e.g., in corn event 5307), a Cry3Bb1 protein (e.g.,in corn event MON88017), a Cry34/35Ab1 binary protein (e.g., in cornevent DAS-59122) and/or a RNAi trait, such as DvSnf7 dsRNA (e.g., incorn event MON87411).

Accordingly, as one aspect, the invention provides a modified Axmi205toxin, wherein said modified Axmi205 toxin has insecticidal activityagainst a plant pest (e.g., an insect pest, such as a coleopteran pest)and comprises a modification (e.g., deletion, substitution and/orinsertion) of one or more amino acids incorporated in the amino acidsequence of SEQ ID NO: 1 or an amino acid sequence that is substantiallyidentical to the amino acid sequence of the polypeptide of SEQ ID NO: 1,and wherein the modification results in enhanced digestion of themodified Axmi205 toxin by a mammalian digestive protease (e.g., pepsin)as compared with an Axmi205 toxin that is identical except that it doesnot comprise the modification.

In other aspects, the modified Axmi205 toxin is toxic to a coleopteraninsect pest, for example, a corn rootworm (e.g., a Diabrotica species),Bean leaf beetle (Cerotoma trifurcata), Colorado Potato Beetle(Leptinotarsa decemlineata) and/or Boll Weevil (Anthonomus grandis).

As a further aspect, the invention provides a modified Axmi205 toxinthat comprises, consists essentially of, or consists of the polypeptideof SEQ ID NO: 1, which has been modified by the modification (e.g.,deletion, substitution and/or insertion) of one or more amino acidsresulting in enhanced digestion by a mammalian digestive protease (e.g.,pepsin).

In further aspects, digestion of the modified Axmi205 toxin is enhanced(faster and/or more complete) by a mammalian digestive protease (e.g.,pepsin) such that there is a lesser amount of fragments above 4 kDaremaining as compared with an Axmi205 toxin that does not comprise themodification (e.g., deletion, substitution and/or insertion), whentested under the same conditions (e.g., enzyme concentration, proteinconcentration, pH, temperature and/or time). For example, as describedin Example 5, digestion with pepsin can be carried out at approximately37° C. and approximately pH 1.2, optionally with an enzyme concentrationof approximately 10 Units (U) pepsin per microgram of protein. Inembodiments, no fragments of the modified Axmi205 toxin above 4 kDa arepresent (e.g., detectable) after 10 minutes of digestion with themammalian digestive protease (e.g., pepsin).

As a further aspect, the modification (deletion, substitution and/orinsertion of one or more amino acids) is in a portion of the polypeptideof SEQ ID NO: 1 from amino acid 402 to amino acid 497 or thecorresponding portion of another Axmi205 toxin.

In some aspects, the invention provides a modified Axmi205 toxincomprising:

-   -   a) one or more amino acids with an aliphatic hydrophobic side        chain are deleted, substituted and/or inserted;    -   b) one or more amino acids with an aromatic hydrophobic side        chain are deleted, substituted and/or inserted;    -   c) one or more amino acids with a polar neutral side chain are        deleted, substituted and/or inserted;    -   d) one or more amino acids with an acidic side chain are        deleted, substituted and/or inserted;    -   e) one or more amino acids with a basic side chain are deleted,        substituted and/or inserted; or    -   f) any combination of (a) to (e).

In further exemplary aspects, the modification incorporated in themodified Axmi205 toxin produces a new protease (e.g., pepsin) cleavagesite. Optionally, according to this aspect, one or more amino acids withan aliphatic hydrophobic side chain and/or an aromatic hydrophobic sidechain are substituted and/or inserted.

In another aspect, the modification incorporated in the modified Axmi205toxin eliminates one or more cysteine residues in the Axmi205 toxin bysubstitution with another amino acid.

According to still further aspects, the invention provides a modifiedAxmi205 toxin comprising:

-   -   a) an amino acid substitution at amino acid K402 in the        polypeptide of SEQ ID NO:1 or the corresponding lysine residue        in another Axmi205 toxin;    -   b) amino acid substitutions at amino acids K402 and Y404 in the        polypeptide of SEQ ID NO:1 or the corresponding lysine and        tyrosine residues in another Axmi205 toxin;    -   c) an amino acid substitution at amino acid C482 in the        polypeptide of SEQ ID NO:1 or the corresponding cysteine residue        in another Axmi205 toxin;    -   d) an amino acid substitution at amino acid C507 in the        polypeptide of SEQ ID NO:1 or the corresponding cysteine residue        in another Axmi205 toxin;    -   e) amino acid substitutions at amino acids M422 and M423 in the        polypeptide of SEQ ID NO:1 or the corresponding methionine        residues in another Axmi205 toxin;    -   f) an amino acid insertion between amino acids A475 and G476 in        the polypeptide of SEQ ID NO:1 or the corresponding alanine and        glycine residues in another Axmi205 toxin;    -   g) an amino acid insertion between amino acids G496 and D497 in        the polypeptide of SEQ ID NO:1 or the corresponding glycine and        aspartic acid residues in another Axmi205 toxin;    -   h) an amino acid insertion between amino acids Q471 and P472 in        the polypeptide of SEQ ID NO:1 or the corresponding glycine and        aspartic acid residues in another Axmi205 toxin;    -   i) an amino acid insertion between amino acids A479 and S480 in        the polypeptide of SEQ ID NO:1 or the corresponding glycine and        aspartic acid residues in another Axmi205 toxin;    -   j) an amino acid insertion between amino acids Y489 and N490 in        the polypeptide of SEQ ID NO:1 or the corresponding glycine and        aspartic acid residues in another Axmi205 toxin;    -   k) a single amino acid insertion within the region bounded by        amino acid positions 469 and 483 of SEQ ID NO: 1; or    -   l) a single amino acid insertion within the region bounded by        amino acid positions 483 and 501 of SEQ ID NO:1.

In yet another aspect, the invention provides a modified Axmi205 toxincomprising:

-   -   a) a substitution of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain at amino acid K402 in the polypeptide of SEQ ID NO:1        or the corresponding lysine in another Axmi205 toxin;    -   b) amino acid substitutions of (i) an amino acid with an        aliphatic hydrophobic side chain, an amino acid with an aromatic        hydrophobic side chain, an amino acid with a polar neutral side        chain, an amino acid with an acidic side chain, or an amino acid        with a basic side chain at amino acid K402 in the polypeptide of        SEQ ID NO:1 or the corresponding lysine residue in another        Axmi205 toxin; and (ii) an amino acid with an aliphatic        hydrophobic side chain, an amino acid with an aromatic        hydrophobic side chain, an amino acid with a polar neutral side        chain, an amino acid with an acidic side chain, or an amino acid        with a basic side chain at amino acid Y404 in the polypeptide of        SEQ ID NO:1 or the corresponding tyrosine residue in another        Axmi205 toxin;    -   c) a substitution of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain at amino acid C482 in the polypeptide of SEQ ID NO:1        or the corresponding cysteine residue in another Axmi205 toxin;    -   d) a substitution of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain at amino acid C507 in the polypeptide of SEQ ID NO:1        or the corresponding cysteine residue in another Axmi205 toxin;    -   e) amino acid substitutions of (i) an amino acid with an        aliphatic hydrophobic side chain, an amino acid with an aromatic        hydrophobic side chain, an amino acid with a polar neutral side        chain, an amino acid with an acidic side chain, or an amino acid        with a basic side chain at amino acid M422 in the polypeptide of        SEQ ID NO:1 or the corresponding methionine residue in another        Axmi205 toxin; and (ii) an amino acid with an aliphatic        hydrophobic side chain, an amino acid with an aromatic        hydrophobic side chain, an amino acid with a polar neutral side        chain, an amino acid with an acidic side chain, or an amino acid        with a basic side chain at amino acid M423 in the polypeptide of        SEQ ID NO:1 or the corresponding methionine residue in another        Axmi205 toxin;    -   f) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain between amino acids A475 and G476 in the polypeptide        of SEQ ID NO:1 or the corresponding alanine and glycine residues        in another Axmi205 toxin;    -   g) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain between amino acids G496 and D497 in the polypeptide        of SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin;    -   h) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain between amino acids Q471 and P472 in the polypeptide        of SEQ ID NO:1 or the corresponding alanine and glycine residues        in another Axmi205 toxin;    -   i) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain between amino acids A479 and S480 in the polypeptide        of SEQ ID NO:1 or the corresponding alanine and glycine residues        in another Axmi205 toxin; or    -   j) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain between amino acids Y489 and N490 in the polypeptide        of SEQ ID NO:1 or the corresponding alanine and glycine residues        in another Axmi205 toxin.

In further illustrative aspects, the invention provides a modifiedAxmi205 toxin comprising:

-   -   a) an amino acid substitution of K402F, K402N or K402D in the        polypeptide of SEQ ID NO:1 or the corresponding lysine in        another Axmi205 toxin;    -   b) amino acid substitutions of (i) K402L and Y404F, or (ii)        K402D and Y404L in the polypeptide of SEQ ID NO:1 or the        corresponding lysine and tyrosine residues in another Axmi205        toxin;    -   c) an amino acid substitution of C482S, C482D or C482F in the        polypeptide of SEQ ID NO:1 or the corresponding cysteine residue        in another Axmi205 toxin;    -   d) an amino acid substitution of C507S, C507L, C507A, C507F,        C507D or C507R in the polypeptide of SEQ ID NO:1 or the        corresponding cysteine residue in another Axmi205 toxin;    -   e) amino acid substitutions of (i) M422S and M423L, (ii) M422T        and M423F, (iii) M422S and M423E, (iv) M422D and M423E, (v)        M422K and M423R, or (vi) M422K and M423F or the corresponding        methionine residues in another Axmi205 toxin;    -   f) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids A475 and G476 in the polypeptide of        SEQ ID NO:1 or the corresponding alanine and glycine residues in        another Axmi205 toxin;    -   g) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids G496 and D497 in the polypeptide of        SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin;    -   h) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids Q471 and P472 in the polypeptide of        SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin;    -   i) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids A479 and S480 in the polypeptide of        SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin; or    -   j) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids Y489 and N490 in the polypeptide of        SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin.

Still further, in representative aspects, the invention provides amodified Axmi205 toxin comprising, consisting essentially of, orconsisting of the amino acid sequence of: SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO:69, SEQ ID NO: 73, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ IDNO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103,SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ IDNO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121,SEQ ID NO: 123, SEQ ID NO: 125, or SEQ ID NO: 127.

As further aspects, the invention provides polynucleotides comprising anucleotide sequence encoding a modified Axmi205 toxin of the invention,optionally codon optimized for expression in a plant. Also provided areexpression cassettes and vectors comprising the polynucleotides of theinvention.

According to some aspects, the invention provides a polynucleotidecomprising a nucleotide sequence that comprises, consists essentiallyof, or consists of:

-   -   a) a nucleotide sequence of SEQ ID NO: 70, SEQ ID NO: 12, SEQ ID        NO: 14, SEQ ID NO: 18, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO:        58, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,        SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ        ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID        NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO:        102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:        110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:        118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, or SEQ ID        NO: 126;    -   b) a nucleotide sequence that is substantially identical to the        nucleotide sequence of (a);    -   c) a nucleotide sequence that anneals under stringent        hybridization conditions to the nucleotide sequence of (a) or        (b); or    -   d) a nucleotide sequence that differs from the nucleotide        sequence of (a), (b) or (c) due to the degeneracy of the genetic        code.

Optionally, according to this aspect, the polynucleotide comprises,consists essentially of, or consists of the nucleotide sequence of SEQID NO: 70, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 44,SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO:76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ IDNO: 84, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102,SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ IDNO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120,SEQ ID NO: 122, SEQ ID NO: 124, or SEQ ID NO: 126.

As a further aspect, the invention provides a transgenic cell (e.g., atransgenic plant cell such as a dicot cell or monocot cell, or atransgenic bacterial cell), transgenic plant part, transgenic plantculture, and transgenic plant seed that comprises a nucleotide sequence,expression cassette, vector and/or insecticidal protein of theinvention.

As still a further aspect, the invention encompasses transgenic plantscomprising a plant cell, plant part, nucleotide sequence, expressioncassette, vector and/or insecticidal protein of the invention.

As a further aspect are seeds that produce the transgenic plants of theinvention and seeds produced by the transgenic plants of the invention.

Also provided are harvested products derived from the transgenic plantsof the invention, wherein the harvested product optionally comprises anucleotide sequence, expression cassette, vector and/or insecticidalprotein of the invention. Further provided are processed productsderived from the harvested products of the invention, wherein theharvested product optionally comprises a nucleotide sequence, expressioncassette, vector and/or insecticidal protein of the invention. Inembodiments, the harvested product or processed product comprises aninsecticidal protein of the invention and has increased resistance to aninsect pest (e.g., a coleopteran insect pest, such as WCRW)).

As still a further aspect, the invention provides an insecticidalcomposition comprising an insecticidal protein of the invention and anagriculturally acceptable carrier.

Still further, the invention provides as an additional aspect a methodof producing a transgenic plant with increased resistance to an insectpest (e.g., a coleopteran insect pest, such as WCRW)). In embodiments,the method comprises introducing into a plant a polynucleotide,expression cassette, or vector of the invention, wherein theinsecticidal protein is expressed in the plant, thereby producing atransgenic plant with increased resistance to an insect pest.Optionally, the introducing step comprises: (i) transforming a plantcell with the polynucleotide, expression cassette or vector andregenerating a transgenic plant; or (ii) crossing a first plantcomprising the polynucleotide, expression cassette or vector with asecond plant. In embodiments, the method further comprises producing aseed from the transgenic plant. In embodiments, the method furthercomprises obtaining a progeny plant from the transgenic plant, whereinthe progeny plant comprises the polynucleotide, the expression cassetteor the vector, expresses the insecticidal protein and has increasedresistance to an insect pest.

As yet another aspect, the invention provides a method of producing atransgenic plant with increased resistance to an insect pest (e.g., acoleopteran insect pest, such as WCRW)), the method comprising: (a)planting a seed comprising a polynucleotide, expression cassette orvector of the invention; and (b) growing a transgenic plant from theseed, wherein the transgenic plant comprises the polynucleotide,expression cassette or vector and produces the insecticidal protein andhas increased resistance to an insect pest. In embodiments, the methodfurther comprises: (c) harvesting a seed from the transgenic plant of(b), wherein the harvested seed comprises the polynucleotide, expressioncassette, vector and/or the insecticidal protein. Optionally, the seedhas increased resistance against an insect pest (e.g., a coleopteraninsect pest, such as WCRW)).

Still further, as another aspect, the invention provides a method ofproducing a seed. In embodiments, the method comprises: (a) providing atransgenic plant that comprises a polynucleotide, expression cassette orvector of the invention; and (b) harvesting a seed from the transgenicplant of (a), wherein the harvested seed comprises the polynucleotide,expression cassette or vector and/or an insecticidal protein of theinvention. Optionally, the seed has increased resistance against aninsect pest (e.g., a coleopteran insect pest, such as WCRW).

The invention further contemplates a method of producing a hybrid plantseed. In representative embodiments, the method comprises: (a) crossinga first inbred plant, which is a transgenic plant comprising apolynucleotide, expression cassette or vector of the invention with adifferent inbred plant, which may or may not comprise a polynucleotide,expression cassette or vector of the invention; and (b) allowing ahybrid seed to form. In embodiments, the hybrid seed comprises apolynucleotide, expression cassette or vector and/or an insecticidalprotein of the invention. Optionally, the seed has increased resistanceagainst an insect pest (e.g., a coleopteran insect pest, such as WCRW)).

As another aspect, the invention provides a method of controlling aninsect pest (e.g., a coleopteran insect pest, such as corn rootworm),the method comprising delivering to the insect pest or an environmentthereof a composition comprising an effective amount of an insecticidalprotein or insecticidal composition of the invention. In embodiments,the method is a method of controlling a coleopteran insect pest (e.g., acorn rootworm, such as WCRW) that is resistant to a mCry3A protein(e.g., in maize event MIR604), a eCry3.1Ab protein (e.g., in maize event5307), a Cry3Bb1 protein (e.g., in corn event MON88017), a Cry34/35Ab1binary protein (e.g., in corn event DAS-59122) and/or a RNAi trait, suchas DvSnf7 dsRNA (e.g., in corn event MON87411).

The invention is also drawn to methods of using the polynucleotides ofthe invention, for example, in DNA constructs or expression cassettes orvectors for transformation and expression in organisms, including plantsand microorganisms, such as bacteria. The nucleotide or amino acidsequences may be native or synthetic sequences that have been designedfor expression in an organism such as a plant or bacteria. The inventionis further drawn to methods of making the insecticidal proteins of theinvention and to methods of using the polynucleotide sequences andinsecticidal proteins, for example in microorganisms to control insectsor in transgenic plants to confer protection from insect damage.

Another aspect of the invention includes insecticidal compositions andformulations comprising the insecticidal proteins of the invention, andmethods of using the compositions or formulations to control insectpopulations, for example by applying the compositions or formulations toinsect-infested areas, or to prophylactically treat insect-susceptibleareas or plants to confer protection against the insect pests.Optionally, the compositions or formulations of the invention may, inaddition to the insecticidal protein of the invention, comprise otherpesticidal agents such as chemical pesticides, other pesticidalproteins, or dsRNA, e.g., in order to augment or enhance theinsect-controlling capability of the composition or formulation and/orfor insect resistance management.

The compositions and methods of the invention are useful for controllinginsect pests that attack plants, particularly crop plants. Thecompositions of the invention are also useful for detecting the presenceof an insecticidal protein or a nucleic acid encoding the same incommercial products or transgenic organisms.

The invention also provides for uses of the insecticidal proteins,nucleic acids, transgenic plants, plant parts, seed and insecticidalcompositions of the invention, for example, to control an insect pest,such as a coleopteran pest (e.g., WCRW).

In embodiments, the invention provides a method of using apolynucleotide, expression cassette, vector or host cell of theinvention to produce an insecticidal composition for controlling aninsect pest (e.g., a coleopteran insect pest, such as WCRW)).

In embodiments, the invention provides a method of using apolynucleotide, expression cassette or vector of the invention toproduce a transgenic seed, where the transgenic seed grows a transgenicplant with increased resistance to an insect pest.

As another aspect, the invention also contemplates the use of atransgenic plant of the invention to produce a transgenic seed, which isoptionally a hybrid seed.

In embodiments, the invention provides a method of using an insecticidalprotein, polynucleotide, expression cassette, vector, transgenic plantor insecticidal composition of the invention to prevent the developmentof resistance in a population of a target coleopteran insect pest to amCry3A protein (e.g., in maize event MIR604), an eCry3.1Ab protein(e.g., in maize event 5307), a Cry3Bb1 protein (e.g., in corn eventMON88017), a Cry34/35Ab1 binary protein (e.g., in corn event DAS-59122)and/or a RNAi trait, such as DvSnf7 dsRNA (e.g., in corn eventMON87411).

These and other features, aspects, and advantages of the invention willbecome better understood with reference to the following detaileddescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Axmi205 sequence showing 2^(nd) domain underlined, predictedpepsin cleavage sites shaded in gray and Cysteine residues in C-terminaldomain in bold-face type.

FIG. 2 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #5 (K402F), #21 (C482S), #23 (C507S), #28 (M4222S &M423L) and #34 (475-Phe-476).

FIG. 3 . Results of SGF assay at times T0 and T5 for wild-type Axmi205and eAxmi205 mutants #36D (496-Asp-497), #37L (471-Leu-472), #38L(479-Leu-480) and #39L (489-Leu-490).

FIG. 4 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #13 (C406S), #16 (C482S), #18 (C445S), #21 (C482S)and #23 (C439S).

FIG. 5 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #1 (K328Y), #9 (R416L), #14 (C406L), #19 (R454F)and #25 (F378L).

FIG. 6 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #26 (S495L), #28 (M4222S & M423L), #31(396-Leu-397), #32 (330-Leu-331), and #35 (367-Leu-368).

FIG. 7 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #3 (K328F), #4 (Y404F), #5 (K402F), #6 (K402N) and#22 (C482L).

FIG. 8 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #2 (K328L), #8 (Y404F & K402L), #33 (456-Leu-457),#34 (475-Leu-476) and #36 (496-Leu-497).

FIG. 9 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #11 (R391L), #12 (R391I) and #20 (R464L).

FIG. 10 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #2 (K402L), #15 (P411L), #17 (C439L), #27 (G496L)and #29 (V467S & S468L).

FIG. 11 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #5D (K402D), #21F (C482F), #23F (C507F) and #28TF(M422T & M423F).

FIG. 12 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #28KR (M422K & M423R), #34D (475-Asp-476), #34F(475-Phe-476) and #34R (475-Arg-476).

FIG. 13 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #23D (C507D), #23L (C507L), #23R (C507R), #28DE(M422D & M423E) and #36D (496-Asp-497).

FIG. 14 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #21D (C482D), #28SE (M4222S & M423E), #36R(496-Arg-497), #37F (471-Phe-472) and #37L (471-Leu-472).

FIG. 15 . Results of SGF assay at times T0 and T10 for wild-type Axmi205and eAxmi205 mutants #28KF (M4222K & M423F), #36F (496-Phe-497), #38F(479-Phe-480), #38L (479-Leu-480), #39F (489-Phe-490), #39L(489-Leu-490).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of the native Axmi205 protein.

SEQ ID NO: 2 is the cDNA sequence of the native Axmi205 protein.

SEQ ID NO: 3 is the amino acid sequence of the eAxmi205 #1 mutantprotein (K328Y).

SEQ ID NO: 4 is a nucleotide sequence encoding the eAxmi205 #1 mutantprotein of SEQ ID NO: 3.

SEQ ID NO: 5 is the amino acid sequence of the eAxmi205 #2 mutantprotein (K328L).

SEQ ID NO: 6 is a nucleotide sequence encoding the eAxmi205 #2 mutantprotein of SEQ ID NO: 5.

SEQ ID NO: 7 is the amino acid sequence of the eAxmi205 #3 mutantprotein (K328F).

SEQ ID NO: 8 is a nucleotide sequence encoding the eAxmi205 #3 mutantprotein of SEQ ID NO: 7.

SEQ ID NO: 9 is the amino acid sequence of the eAxmi205 #4 mutantprotein (Y404F).

SEQ ID NO: 10 is a nucleotide sequence encoding the eAxmi205 #4 mutantprotein of SEQ ID NO: 9.

SEQ ID NO: 11 is the amino acid sequence of the eAxmi205 #5 mutantprotein (K402F).

SEQ ID NO: 12 is a nucleotide sequence encoding the eAxmi205 #5 mutantprotein of SEQ ID NO: 11.

SEQ ID NO: 13 is the amino acid sequence of the eAxmi205 #6 mutantprotein (K402N).

SEQ ID NO: 14 is a nucleotide sequence encoding the eAxmi205 #6 mutantprotein of SEQ ID NO: 13.

SEQ ID NO: 15 is the amino acid sequence of the eAxmi205 #7 mutantprotein (K402L).

SEQ ID NO: 16 is a nucleotide sequence encoding the eAxmi205 #7 mutantprotein of SEQ ID NO: 15.

SEQ ID NO: 17 is the amino acid sequence of the eAxmi205 #8 mutantprotein (Y404F+K402L).

SEQ ID NO: 18 is a nucleotide sequence encoding the eAxmi205 #8 mutantprotein of SEQ ID NO: 17.

SEQ ID NO: 19 is the amino acid sequence of the eAxmi205 #9 mutantprotein (R416L).

SEQ ID NO: 20 is a nucleotide sequence encoding the eAxmi205 #9 mutantprotein of SEQ ID NO: 19.

SEQ ID NO: 21 is the amino acid sequence of the eAxmi205 #10 mutantprotein (P386L).

SEQ ID NO: 22 is a nucleotide sequence encoding the eAxmi205 #10 mutantprotein of SEQ ID NO: 21.

SEQ ID NO: 23 is the amino acid sequence of the eAxmi205 #11 mutantprotein (R391L).

SEQ ID NO: 24 is a nucleotide sequence encoding the eAxmi205 #11 mutantprotein of SEQ ID NO: 23.

SEQ ID NO: 25 is the amino acid sequence of the eAxmi205 #12 mutantprotein (R391I).

SEQ ID NO: 26 is a nucleotide sequence encoding the eAxmi205 #12 mutantprotein of SEQ ID NO: 25.

SEQ ID NO: 27 is the amino acid sequence of the eAxmi205 #13 mutantprotein (C406S).

SEQ ID NO: 28 is a nucleotide sequence encoding the eAxmi205 #13 mutantprotein of SEQ ID NO: 27.

SEQ ID NO: 29 is the amino acid sequence of the eAxmi205 #14 mutantprotein (C406L).

SEQ ID NO: 30 is a nucleotide sequence encoding the eAxmi205 #14 mutantprotein of SEQ ID NO: 29.

SEQ ID NO: 31 is the amino acid sequence of the eAxmi205 #15 mutantprotein (P411L).

SEQ ID NO: 32 is a nucleotide sequence encoding the eAxmi205 #15 mutantprotein of SEQ ID NO: 31.

SEQ ID NO: 33 is the amino acid sequence of the eAxmi205 #16 mutantprotein (C439S).

SEQ ID NO: 34 is a nucleotide sequence encoding the eAxmi205 #16 mutantprotein of SEQ ID NO: 33.

SEQ ID NO: 35 is the amino acid sequence of the eAxmi205 #17 mutantprotein (C439L).

SEQ ID NO: 36 is a nucleotide sequence encoding the eAxmi205 #17 mutantprotein of SEQ ID NO: 35.

SEQ ID NO: 37 is the amino acid sequence of the eAxmi205 #18 mutantprotein (C445S).

SEQ ID NO: 38 is a nucleotide sequence encoding the eAxmi205 #18 mutantprotein of SEQ ID NO: 37.

SEQ ID NO: 39 is the amino acid sequence of the eAxmi205 #19 mutantprotein (R454F).

SEQ ID NO: 40 is a nucleotide sequence encoding the eAxmi205 #19 mutantprotein of SEQ ID NO: 39.

SEQ ID NO: 41 is the amino acid sequence of the eAxmi205 #20 mutantprotein (R464L).

SEQ ID NO: 42 is a nucleotide sequence encoding the eAxmi205 #20 mutantprotein of SEQ ID NO: 41.

SEQ ID NO: 43 is the amino acid sequence of the eAxmi205 #21 mutantprotein (C482S).

SEQ ID NO: 44 is a nucleotide sequence encoding the eAxmi205 #21 mutantprotein of SEQ ID NO: 43.

SEQ ID NO: 45 is the amino acid sequence of the eAxmi205 #22 mutantprotein (C482L).

SEQ ID NO: 46 is a nucleotide sequence encoding the eAxmi205 #22 mutantprotein of SEQ ID NO: 45.

SEQ ID NO: 47 is the amino acid sequence of the eAxmi205 #23 mutantprotein (C507S).

SEQ ID NO: 48 is a nucleotide sequence encoding the eAxmi205 #23 mutantprotein of SEQ ID NO: 47.

SEQ ID NO: 49 is the amino acid sequence of the eAxmi205 #24 mutantprotein (C406S+C439S+C445S+C482S+C507S).

SEQ ID NO: 50 is a nucleotide sequence encoding the eAxmi205 #24 mutantprotein of SEQ ID NO: 49.

SEQ ID NO: 51 is the amino acid sequence of the eAxmi205 #25 mutantprotein (F378L).

SEQ ID NO: 52 is a nucleotide sequence encoding the eAxmi205 #25 mutantprotein of SEQ ID NO: 51.

SEQ ID NO: 53 is the amino acid sequence of the eAxmi205 #26 mutantprotein (S495L).

SEQ ID NO: 54 is a nucleotide sequence encoding the eAxmi205 #26 mutantprotein of SEQ ID NO: 53.

SEQ ID NO: 55 is the amino acid sequence of the eAxmi205 #27 mutantprotein (G496L).

SEQ ID NO: 56 is a nucleotide sequence encoding the eAxmi205 #27 mutantprotein of SEQ ID NO: 55.

SEQ ID NO: 57 is the amino acid sequence of the eAxmi205 #28 mutantprotein (M422S+M423L).

SEQ ID NO: 58 is a nucleotide sequence encoding the eAxmi205 #28 mutantprotein of SEQ ID NO: 57.

SEQ ID NO: 59 is the amino acid sequence of the eAxmi205 #29 mutantprotein (V467S+S468L).

SEQ ID NO: 60 is a nucleotide sequence encoding the eAxmi205 #29 mutantprotein of SEQ ID NO: 59.

SEQ ID NO: 61 is the amino acid sequence of the eAxmi205 #30 mutantprotein (V467S+S468L+W470G).

SEQ ID NO: 62 is a nucleotide sequence encoding the eAxmi205 #30 mutantprotein of SEQ ID NO: 61.

SEQ ID NO: 63 is the amino acid sequence of the eAxmi205 #31 mutantprotein (396-Leu-397).

SEQ ID NO: 64 is a nucleotide sequence encoding the eAxmi205 #31 mutantprotein of SEQ ID NO: 63.

SEQ ID NO: 65 is the amino acid sequence of the eAxmi205 #32 mutantprotein (330-Leu-331).

SEQ ID NO: 66 is a nucleotide sequence encoding the eAxmi205 #32 mutantprotein of SEQ ID NO: 65.

SEQ ID NO: 67 is the amino acid sequence of the eAxmi205 #33 mutantprotein (456-Leu-457).

SEQ ID NO: 68 is a nucleotide sequence encoding the eAxmi205 #33 mutantprotein of SEQ ID NO: 67.

SEQ ID NO: 69 is the amino acid sequence of the eAxmi205 #34 mutantprotein (475-Leu-476).

SEQ ID NO: 70 is a nucleotide sequence encoding the eAxmi205 #34 mutantprotein of SEQ ID NO: 69.

SEQ ID NO: 71 is the amino acid sequence of the eAxmi205 #35 mutantprotein (367-Leu-368).

SEQ ID NO: 72 is a nucleotide sequence encoding the eAxmi205 #35 mutantprotein of SEQ ID NO: 71.

SEQ ID NO: 73 is the amino acid sequence of the eAxmi205 #36 mutantprotein (496-Leu-497).

SEQ ID NO: 74 is a nucleotide sequence encoding the eAxmi205 #36 mutantprotein of SEQ ID NO: 73.

SEQ ID NO: 75 is a maize optimized nucleotide sequence encoding theeAxmi205 #23 mutant protein of SEQ ID NO: 47.

SEQ ID NO: 76 is a maize optimized nucleotide sequence encoding theeAxmi205 #28 mutant protein of SEQ ID NO: 57.

SEQ ID NO: 77 is a maize optimized nucleotide sequence encoding theeAxmi205 #34 mutant protein of SEQ ID NO: 69.

SEQ ID NO: 78 is a nucleotide sequence encoding the eAxmi205 #5D mutantprotein of SEQ ID NO: 79.

SEQ ID NO: 79 is the amino acid sequence of the eAxmi205 #5D mutantprotein (K402D).

SEQ ID NO: 80 is a nucleotide sequence encoding the eAxmi205 #21F mutantprotein of SEQ ID NO: 81.

SEQ ID NO: 81 is the amino acid sequence of the eAxmi205 #21F mutantprotein (C482F).

SEQ ID NO: 82 is a nucleotide sequence encoding the eAxmi205 #21D mutantprotein of SEQ ID NO: 83.

SEQ ID NO: 83 is the amino acid sequence of the eAxmi205 #21D mutantprotein (C482D).

SEQ ID NO: 84 is a nucleotide sequence encoding the eAxmi205 #23L mutantprotein of SEQ ID NO: 85.

SEQ ID NO: 85 is the amino acid sequence of the eAxmi205 #23L mutantprotein (C507L).

SEQ ID NO: 86 is a nucleotide sequence encoding the eAxmi205 #23A mutantprotein of SEQ ID NO: 87.

SEQ ID NO: 87 is the amino acid sequence of the eAxmi205 #23A mutantprotein (C507A).

SEQ ID NO: 88 is a nucleotide sequence encoding the eAxmi205 #23F mutantprotein of SEQ ID NO: 89.

SEQ ID NO: 89 is the amino acid sequence of the eAxmi205 #23F mutantprotein (C507F).

SEQ ID NO: 90 is a nucleotide sequence encoding the eAxmi205 #23D mutantprotein of SEQ ID NO: 91.

SEQ ID NO: 91 is the amino acid sequence of the eAxmi205 #23D mutantprotein (C507D).

SEQ ID NO: 92 is a nucleotide sequence encoding the eAxmi205 #23R mutantprotein of SEQ ID NO: 93.

SEQ ID NO: 93 is the amino acid sequence of the eAxmi205 #23R mutantprotein (C507R).

SEQ ID NO: 94 is a nucleotide sequence encoding the eAxmi205 #28TFmutant protein of SEQ ID NO: 95.

SEQ ID NO: 95 is the amino acid sequence of the eAxmi205 #28TF mutantprotein (M422T+M423F).

SEQ ID NO: 96 is a nucleotide sequence encoding the eAxmi205 #28DEmutant protein of SEQ ID NO: 97.

SEQ ID NO: 97 is the amino acid sequence of the eAxmi205 #28DE mutantprotein (M422D+M423E).

SEQ ID NO: 98 is a nucleotide sequence encoding the eAxmi205 #28KRmutant protein of SEQ ID NO: 99.

SEQ ID NO: 99 is the amino acid sequence of the eAxmi205 #28KR mutantprotein (M422K+M423R).

SEQ ID NO: 100 is a nucleotide sequence encoding the eAxmi205 #28SEmutant protein of SEQ ID NO: 101.

SEQ ID NO: 101 is the amino acid sequence of the eAxmi205 #28SE mutantprotein (M422S+M423E).

SEQ ID NO: 102 is a nucleotide sequence encoding the eAxmi205 #28KFmutant protein of SEQ ID NO: 103.

SEQ ID NO: 103 is the amino acid sequence of the eAxmi205 #28KF mutantprotein (M422K+M423F).

SEQ ID NO: 104 is a nucleotide sequence encoding the eAxmi205 #34Fmutant protein of SEQ ID NO: 105.

SEQ ID NO: 105 is the amino acid sequence of the eAxmi205 #34F mutantprotein (475-Phe-476).

SEQ ID NO: 106 is a nucleotide sequence encoding the eAxmi205 #34Dmutant protein of SEQ ID NO: 107.

SEQ ID NO: 107 is the amino acid sequence of the eAxmi205 #34D mutantprotein (475-Asp-476).

SEQ ID NO: 108 is a nucleotide sequence encoding the eAxmi205 #34Rmutant protein of SEQ ID NO: 109.

SEQ ID NO: 109 is the amino acid sequence of the eAxmi205 #34R mutantprotein (475-Arg-476).

SEQ ID NO: 110 is a nucleotide sequence encoding the eAxmi205 #36Dmutant protein of SEQ ID NO: 111.

SEQ ID NO: 111 is the amino acid sequence of the eAxmi205 #36D mutantprotein (496-Asp-497).

SEQ ID NO: 112 is a nucleotide sequence encoding the eAxmi205 #36Fmutant protein of SEQ ID NO: 113.

SEQ ID NO: 113 is the amino acid sequence of the eAxmi205 #36F mutantprotein (496-Phe-497).

SEQ ID NO: 114 is a nucleotide sequence encoding the eAxmi205 #36Rmutant protein of SEQ ID NO: 115.

SEQ ID NO: 115 is the amino acid sequence of the eAxmi205 #36R mutantprotein (496-Arg-497).

SEQ ID NO: 116 is a nucleotide sequence encoding the eAxmi205 #37Fmutant protein of SEQ ID NO: 117.

SEQ ID NO: 117 is the amino acid sequence of the eAxmi205 #37F mutantprotein (471-Phe-472).

SEQ ID NO: 118 is a nucleotide sequence encoding the eAxmi205 #37Lmutant protein of SEQ ID NO: 119.

SEQ ID NO: 119 is the amino acid sequence of the eAxmi205 #37L mutantprotein (471-Leu-472).

SEQ ID NO: 120 is a nucleotide sequence encoding the eAxmi205 #38Fmutant protein of SEQ ID NO: 121.

SEQ ID NO: 121 is the amino acid sequence of the eAxmi205 #38F mutantprotein (479-Phe-480).

SEQ ID NO: 122 is a nucleotide sequence encoding the eAxmi205 #38Lmutant protein of SEQ ID NO: 123.

SEQ ID NO: 123 is the amino acid sequence of the eAxmi205 #38L mutantprotein (479-Leu-480).

SEQ ID NO: 124 is a nucleotide sequence encoding the eAxmi205 #39Fmutant protein of SEQ ID NO: 125.

SEQ ID NO: 125 is the amino acid sequence of the eAxmi205 #39F mutantprotein (489-Phe-490).

SEQ ID NO: 126 is a nucleotide sequence encoding the eAxmi205 #39Lmutant protein of SEQ ID NO: 127.

SEQ ID NO: 127 is the amino acid sequence of the eAxmi205 #39L mutantprotein (489-Leu-490).

DETAILED DESCRIPTION

This description is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. Thus, theinvention contemplates that in some embodiments of the invention, anyfeature or combination of features set forth herein can be excluded oromitted. In addition, numerous variations and additions to the variousembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention. Hence, the following descriptions are intended toillustrate some particular embodiments of the invention, and not toexhaustively specify all permutations, combinations and variationsthereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties for theteachings relevant to the sentence and/or paragraph in which thereference is presented.

Nucleotide sequences provided herein are presented in the 5′ to 3′direction, from left to right and are presented using the standard codefor representing nucleotide bases as set forth in 37 CFR §§ 1.821-1.825and the World Intellectual Property Organization (WIPO) Standard ST.25,for example: adenine (A), cytosine (C), thymine (T), and guanine (G).

Amino acids are likewise indicated using the WIPO Standard ST.25, forexample: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N),aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamicacid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile;1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M),phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a composition comprises components A, Band C, it is specifically intended that any of A, B or C, or acombination thereof, can be omitted and disclaimed singularly or in anycombination.

Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount. As used herein, phrases such as “between about X andY” mean “between about X and about Y” and phrases such as “from about Xto Y” mean “from about X to about Y.”

As used herein, phrases such as “between about X and Y”, “between aboutX and about Y”, “from X to Y” and “from about X to about Y” (and similarphrases) should be interpreted to include X and Y, unless the contextindicates otherwise.

By “activity” of an insecticidal protein of the invention is meant thatthe insecticidal protein functions as an orally active insect controlagent, has a toxic effect, for example, by inhibiting the ability of theinsect pest to survive, grow, and/or reproduce (e.g., causing morbidityand/or mortality) and/or is able to disrupt and/or deter insect feeding,which may or may not cause death of the insect. Thus, when aninsecticidal protein of the invention is delivered to the insect, theresult is typically morbidity and/or mortality of the insect and/or theinsect reduces or stops feeding upon the source that makes theinsecticidal protein available to the insect.

A “coding sequence” is a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Inembodiments, the RNA is then translated to produce a protein.

As used herein, a “codon optimized” nucleotide sequence means anucleotide sequence of a recombinant, transgenic, or syntheticpolynucleotide wherein the codons are chosen to reflect the particularcodon bias that a host cell or organism may have. This is typically donein such a way so as to preserve the amino acid sequence of thepolypeptide encoded by the codon optimized nucleotide sequence. Incertain embodiments, a nucleotide sequence is codon optimized for thecell (e.g., an animal, plant, fungal or bacterial cell) in which theconstruct is to be expressed. For example, a construct to be expressedin a plant cell can have all or parts of its sequence codon optimizedfor expression in a plant. See, for example, U.S. Pat. No. 6,121,014. Inembodiments, the polynucleotides of the invention are codon-optimizedfor expression in a plant cell (e.g., a dicot cell or a monocot cell) orbacterial cell.

To “control” an insect pest means to inhibit, through a toxic effect,the ability of the insect pest to survive, grow, feed and/or reproduceand/or to limit insect-related damage or loss in a crop plant caused bythe insect pest and/or to protect the yield potential of a crop causedby the pest when grown in the presence of an insect pest. To “control”an insect pest may or may not mean killing the insect, although inembodiments of the invention, “control” of the insect means killing theinsect.

The term “comprise”, “comprises” or “comprising,” when used in thisspecification, indicates the presence of the stated features, integers,steps, operations, elements, or components, but does not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) means that the scope of a claim is to beinterpreted to encompass the specified materials or steps recited in theclaim “and those that do not materially alter the basic and novelcharacteristic(s)” of the claimed invention. Thus, the term “consistingessentially of” when used in a claim of this invention is not intendedto be interpreted to be equivalent to “comprising.”

In the context of the invention, “corresponding to” or “corresponds to”means that when the amino acid sequences of modified or homolog proteinsare aligned with each other, the amino acids that “correspond to”certain enumerated positions in the modified or homolog protein arethose that align with these positions in a reference protein, but arenot necessarily in the same exact numerical positions relative to theparticular reference amino acid sequence of the invention. For example,if SEQ ID NO: 1 (native Axmi205) is the reference sequence and isaligned with SEQ ID NO: 73 (Axmi205-36 mutant), the sequence of aminoacid residues 498 to 537 of SEQ ID NO: 73 (immediately following theleucine insertion in mutant Axmi205-36) “corresponds to” amino acidresidues 497 to 536 of SEQ ID NO: 1 (native Axmi205).

As used herein, the term “Cry protein” means an insecticidal protein ofa Bacillus thuringiensis crystal delta-endotoxin type. The term “Cryprotein” can refer to the protoxin form or any insecticidally activefragment or toxin thereof including partially processed and the maturetoxin form (e.g., without the N-terminal peptidyl fragment and/or theC-terminal protoxin tail).

As used herein, to “deliver” or “delivering” (and grammaticalvariations) a composition or insecticidal protein means that thecomposition or insecticidal protein comes in contact with an insect,which facilitates the oral ingestion of the composition or insecticidalprotein, resulting in a toxic effect and control of the insect. Thecomposition or insecticidal protein can be delivered in many recognizedways, including but not limited to, by transgenic plant expression, aformulated protein composition(s), a sprayable protein composition(s), abait matrix, or any other art-recognized protein delivery system.

The term “domain” refers to a set of amino acids conserved at specificpositions along an alignment of sequences of evolutionarily relatedproteins. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely important in the structure,stability and/or function of a protein. Identified by their high degreeof conservation in aligned sequences of a family of protein homologues,they can be used as identifiers to determine if any polypeptide inquestion belongs to a previously identified polypeptide group.

“Effective insect-controlling amount” means that concentration of aninsecticidal protein that inhibits, through a toxic effect, the abilityof an insect to survive, grow, feed and/or reproduce and/or that limitsinsect-related damage or loss in a crop plant. An “effectiveinsect-controlling amount” may or may not mean killing the insect,although in embodiments it indicates killing the insect.

“Expression cassette” as used herein means a nucleic acid moleculecapable of directing expression of at least one polynucleotide ofinterest, such as a polynucleotide that encodes an insecticidal proteinof the invention, in an appropriate host cell, comprising a promoteroperably linked to the polynucleotide of interest which is operablylinked to a termination signal. An “expression cassette” also typicallycomprises additional polynucleotides to facilitate proper translation ofthe polynucleotide of interest. The expression cassette may alsocomprise other polynucleotides not related to the expression of apolynucleotide of interest but which are present due to convenientrestriction sites for removal of the cassette from an expression vector.In embodiments, at least one of the components in the expressioncassette may be heterologous (i.e., foreign) with respect to at leastone of the other components (e.g., a heterologous promoter operativelyassociated with a polynucleotide of interest). The expression cassettemay also be one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression. Typically, however,the expression cassette is heterologous with respect to the host, i.e.,the expression cassette (or even the polynucleotide of interest) doesnot occur naturally in the host cell and has been introduced into thehost cell or an ancestor cell thereof by a transformation process or abreeding process. The expression of the polynucleotide(s) of interest inthe expression cassette is generally under the control of a promoter. Inthe case of a multicellular organism, such as a plant, the promoter canalso be specific or preferential to a particular tissue, or organ, orstage of development (as described in more detail herein). An expressioncassette, or fragment thereof, can also be referred to as “insertedpolynucleotide” or “insertion polynucleotide” when transformed into aplant.

A “gene” is defined herein as a hereditary unit comprising one or morepolynucleotides that occupies a specific location on a chromosome orplasmid and that contains the genetic instruction for a particularcharacteristic or trait in an organism.

As used herein, a “gut protease” or “digestive protease” refers to aprotease naturally found in the digestive tract of an animal (e.g., aninsect or a mammal, such as a human). In embodiments, the “gut protease”or “digestive protease” is from a mammalian (e.g., human). The proteaseis usually involved in the digestion of ingested proteins. Examples ofgut proteases include trypsin, which typically cleaves peptides on theC-terminal side of lysine (K) or arginine (R) residues, andchymotrypsin, which typically cleaves peptides on the C-terminal side ofphenylalanine (F), tryptophan (W) or tyrosine (Y). Pepsin typicallycleaves between two hydrophobic residues.

As used herein, the term “heterologous” means foreign, exogenous,non-native and/or non-naturally occurring. In embodiments, a“heterologous” polynucleotide or polypeptide is a polynucleotide orpolypeptide that is not naturally associated with a host cell into whichit is introduced, including non-naturally occurring multiple copies of anaturally occurring nucleotide sequence or polypeptide. In embodiments,a nucleotide sequence is heterologous to another sequence with which itis operatively associated, e.g., a promoter may be heterologous (i.e.,foreign) to an operatively associated coding sequence.

As used here, “homologous” means native. For example, a homologousnucleotide sequence or amino acid sequence is a nucleotide sequence oramino acid sequence naturally associated with a host cell into which itis introduced, a homologous promoter sequence is the promoter sequencethat is naturally associated with a coding sequence, and the like.

The terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,”“enhancing,” and “enhancement” (and grammatical variations thereof) andsimilar terms, as used herein, describe an elevation and/or improvementin the specified parameter. Thus, in embodiments, the terms “increase,”“increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and“enhancement” (and grammatical variations thereof), and similar termscan indicate an elevation of at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,125%, 150%, 200%, 300%, 400%, 500% or more as compared to a suitablecontrol.

The terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,”“enhancing,” and “enhancement” (and grammatical variations thereof) andsimilar terms, as used herein with respect to the digestion (e.g.,cleavage) of a modified Axmi205 protein of the invention by a digestiveprotease refers to an elevation or improvement in the speed and/orextent of the digestion of the modified Axmi205 toxin. This increase indigestion can be with reference to the level of digestion observed witha suitable control (e.g., the Axmi205 protein of SEQ ID NO: 1 and/or anAxmi205 toxin that is identical to the modified Axmi205 protein of theinvention with the exception that it lacks the modifications of thepresent invention) when tested under the same conditions. For example,as described in Example 5, digestion with pepsin can be carried out atapproximately 37° C. and approximately pH 1.2, optionally, with anenzyme concentration of approximately 10 Units (U) pepsin per microgramof protein. Thus, in embodiments, the terms “increase,” “increasing,”“increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (andgrammatical variations thereof), indicate that protease digestion of themodified Axmi205 toxin is elevated and/or faster by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or moreand/or is more complete (e.g., a lesser amount of undigested orpartially digested fragments remain, optionally at a specified timepoint, e.g., after about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55 or 60 minutes of digestion) as compared with thesuitable control. In embodiments, no detectable fragments (e.g.,immunoreactive fragments) of the modified Axmi205 toxin of the inventionremain above approximately 4 kDa after about 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes digestionwith the digestive protease (e.g., pepsin), optionally under conditionsas defined herein (e.g., approximately 37° C. and approximately pH 1.2,optionally at an enzyme concentration of 10 Units (U)/μg protein).Methods of detecting undigested or partially digested fragments of themodified Axmi205 toxin can be done using any suitable method (e.g.,SDS-PAGE), and immunoreactive fragments can be detected with a suitableantibody (e.g., directed against the Axmi205 toxin of SEQ ID NO: 1), forexample, as described in the working Examples.

The terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,”“enhancing,” and “enhancement” (and grammatical variations thereof) andsimilar terms, as used herein with respect to the level of control of aplant pest describe an elevation in the control of the plant pest, e.g.,by contacting a plant with a polypeptide of the invention (such as, forexample, by transgenic expression or by topical application methods).

This increase in control can be in reference to the level of control ofthe plant pest in the absence of the polypeptide of the invention (e.g.,a plant that is not transgenically expressing the polypeptide or is nottopically treated with the polypeptide). Thus, in embodiments, the terms“increase,” “increasing,” “increased,” “enhance,” “enhanced,”“enhancing,” and “enhancement” (and grammatical variations thereof), andsimilar terms can indicate an elevation of at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more as comparedto a suitable control (e.g., a plant, plant part, plant cell that is notcontacted with a polypeptide of the invention).

“Insecticidal” as used herein is defined as a toxic biological activitycapable of controlling an insect pest, optionally but preferably bykilling them.

A nucleic acid sequence is “isocoding” with a reference nucleic acidsequence when the nucleic acid sequence encodes a polypeptide having thesame amino acid sequence as the polypeptide encoded by the referencenucleic acid sequence.

In representative embodiments, the nucleic acids molecules,polynucleotides or proteins of the invention are “isolated.” An“isolated” nucleic acid molecule, polynucleotide or protein, and thelike, is a nucleic acid molecule, polynucleotide or protein, and thelike that no longer exists in its natural environment. An isolatednucleic acid molecule, polynucleotide or protein of the invention mayexist in a purified form or may exist in a recombinant host such as in atransgenic bacteria or a transgenic plant. In embodiments, an isolatednucleic acid molecule, nucleotide sequence or polypeptide exists in apurified form that is at least partially separated from at least some ofthe other components of the naturally occurring organism or virus, forexample, the cell or viral structural components or other polypeptidesor nucleic acids commonly found associated with the polynucleotide. Inother embodiments, an “isolated” nucleic acid molecule, nucleotidesequence or polypeptide may exist in a non-native environment such as,for example, a recombinant host cell. Thus, for example, with respect tonucleotide sequences, the term “isolated” can mean that the nucleotidesequence is separated from the chromosome and/or cell in which itnaturally occurs. A polynucleotide is also isolated if it is separatedfrom the chromosome and/or cell in which it naturally occurs in and isthen inserted into a genetic context, a chromosome and/or a cell inwhich it does not naturally occur (e.g., a different host cell,different regulatory sequences, and/or different position in the genomethan as found in nature). Accordingly, recombinant nucleic acidmolecules, nucleotide sequences and their encoded polypeptides are“isolated” in that, by the hand of man, they exist apart from theirnative environment and therefore are not products of nature, however, insome embodiments, they can be introduced into and exist in a recombinanthost cell. In representative embodiments, the isolated nucleic acidmolecule, the isolated nucleotide sequence and/or the isolatedpolypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or more pure.

The term “motif” or “consensus sequence” or “signature” refers to ashort conserved region in the sequence of evolutionarily relatedproteins. Motifs are frequently highly conserved parts of domains, butmay also include only part of the domain, or be located outside ofconserved domain (if all of the amino acids of the motif fall outside ofa defined domain).

A “native” or “wild type” nucleic acid, nucleotide sequence, polypeptideor amino acid sequence refers to a naturally occurring or endogenousnucleic acid, nucleotide sequence, polypeptide or amino acid sequence.Thus, for example, a “wild type mRNA” is an mRNA that is naturallyoccurring in or endogenous to the organism.

The terms “nucleic acid,” “nucleic acid molecule,” “nucleotidesequence,” “oligonucleotide” and “polynucleotide” are usedinterchangeably herein, unless the context indicates otherwise, andrefer to a heteropolymer of nucleotides. These terms include withoutlimitation DNA and RNA molecules, including cDNA, genomic DNA, synthetic(e.g., chemically synthesized) DNA and RNA, plasmid DNA, mRNA,anti-sense RNA, and RNA/DNA hybrids, any of which can be linear orbranched, single stranded or double stranded, or a combination thereof.When dsRNA is produced synthetically, less common bases, such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others canalso be used for antisense, dsRNA, and ribozyme pairing. For example,polynucleotides that contain C-5 propyne analogues of uridine andcytidine have been shown to bind RNA with high affinity and to be potentantisense inhibitors of gene expression. Other modifications, such asmodification to the phosphodiester backbone, or the 2′-hydroxy in theribose sugar group of the RNA can also be made. In embodiments, the“nucleic acid,” “nucleic acid molecule,”, “nucleotide sequence,”,“oligonucleotide” or “polynucleotide” refer to DNA.

By “operably linked” or “operably associated” as used herein, it ismeant that the indicated elements are functionally related to eachother, and are also generally physically related. Thus, the term“operably linked” or “operably associated” as used herein, refers tonucleotide sequences on a single nucleic acid molecule that arefunctionally associated. Thus, a first nucleotide sequence that isoperably linked to a second nucleotide sequence, means a situation whenthe first nucleotide sequence is placed in a functional relationshipwith the second nucleotide sequence. For instance, a promoter isoperably associated with a nucleotide sequence if the promoter effectsthe transcription or expression of said nucleotide sequence. Thoseskilled in the art will appreciate that the control sequences (e.g.,promoter) need not be contiguous with the nucleotide sequence to whichit is operably associated, as long as the control sequences function todirect the expression thereof. Thus, for example, interveninguntranslated, yet transcribed, sequences can be present between apromoter and a nucleotide sequence, and the promoter can still beconsidered “operably linked” to or “operatively associated” with thenucleotide sequence.

A “plant” as used herein, refers to any plant at any stage ofdevelopment.

Any plant (or groupings of plants, for example, into a genus or higherorder classification) can be employed in practicing the presentinvention including angiosperms or gymnosperms, monocots or dicots.

Exemplary plants include, but are not limited to corn (Zea mays), canola(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice(Oryza sativa, including without limitation Indica and/or Japonicavarieties), rape (Brassica napus), rye (Secale cereale), sorghum(Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tobacum),potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypiumhirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta),coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananascomosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea(Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig(Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive(Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), apple (Malus pumila),blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape(Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach(Prunus persica), plum (Prunus domestica), pear (Pyrus communis),watermelon (Citrullus vulgaris), duckweed (Lemna spp.), oats (Avenasativa), barley (Hordium vulgare), vegetables, ornamentals, conifers,and turfgrasses (e.g., for ornamental, recreational or forage purposes),and biomass grasses (e.g., switchgrass and miscanthus).

Vegetables include without limitation Solanaceous species (e.g.,tomatoes; Lycopersicon esculentum), lettuce (e.g., Lactuca sativa),carrots (Caucus carota), cauliflower (Brassica oleracea), celery (Apiumgraveolens), eggplant (Solanum melongena), asparagus (Asparagusofficinalis), ochra (Abelmoschus esculentus), green beans (Phaseolusvulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.),members of the genus Cucurbita such as hubbard squash (C. hubbard),butternut squash (C. moschata), zucchini (C. pepo), crookneck squash (C.crookneck), C. argyrosperma, C. argyrosperma ssp sororia, C. digitata,C. ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii, andmembers of the genus Cucumis such as cucumber (Cucumis sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo).

Ornamentals include without limitation azalea (Rhododendron spp.),hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis),roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.),petunias (Petunia hybrida), carnation (Dianthus caryophyllus),poinsettia (Euphorbia pulcherima), and chrysanthemum.

Conifers, which may be employed in practicing the present invention,include, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus effiotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Turfgrass include but are not limited tozoysiagrasses, bentgrasses, fescue grasses, bluegrasses, St.Augustinegrasses, bermudagrasses, bufallograsses, ryegrasses, andorchardgrasses.

Also included are plants that serve primarily as laboratory models,e.g., Arabidopsis.

A “plant cell” is a structural and physiological unit of a plant,comprising a protoplast and a cell wall. The plant cell may be in theform of an isolated single cell or a cultured cell, or as a part of ahigher organized unit such as, for example, plant tissue, a plant organ,or a whole plant. In embodiments, the plant cell is non-propagatingand/or cannot regenerate a whole plant.

A “plant cell culture” means a culture of plant units such as, forexample, protoplasts, cell culture cells, cells in plant tissues,pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos atvarious stages of development.

“Plant material” refers to leaves, stems, roots, flowers or flowerparts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell ortissue cultures, or any other part or product of a plant.

A “plant organ” is a distinct and visibly structured and differentiatedpart of a plant such as a root, stem, leaf, flower bud, or embryo.

As used herein, the term “plant part” includes but is not limited toembryos, pollen, ovules, seeds, leaves, flowers, branches, fruit,stalks, roots, root tips, anthers, and/or plant cells including plantcells that are intact in plants and/or parts of plants, plantprotoplasts, plant tissues, plant cell tissue cultures, plant calli,plant clumps, and the like.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural or functional units. The use ofthis term in conjunction with, or in the absence of, any specific typeof plant tissue as listed above or otherwise embraced by this definitionis not intended to be exclusive of any other type of plant tissue.

A “polynucleotide of interest” refers to any polynucleotide which, whentransferred to an organism, e.g., a plant, confers upon the organism adesired characteristic such as insect resistance, disease resistance,herbicide tolerance, antibiotic resistance, improved nutritional value,improved performance in an industrial process, production of acommercially valuable enzyme or metabolite, an altered reproductivecapability, and the like.

A “portion” or “fragment” of a polypeptide of the invention will beunderstood to mean an amino acid sequence of reduced length relative toa reference amino acid sequence of a polypeptide of the invention. Sucha portion or fragment according to the invention may be, whereappropriate, included in a larger polypeptide of which it is aconstituent (e.g., a tagged or fusion protein). In embodiments, the“portion” or “fragment” substantially retains insecticidal activity(e.g., at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or even 100% ofthe activity of the full-length protein, or has even greaterinsecticidal activity than the full-length protein).

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

The term “promoter” as used herein refers to a polynucleotide, typicallyupstream (5′) of a coding polynucleotide, which controls the expressionof the coding polynucleotide by providing the recognition for RNApolymerase and other transcriptional machinery.

A “protoplast” as used herein, refers to an isolated plant cell withouta cell wall or with only parts of the cell wall.

As used herein, the term “recombinant” refers to a form of nucleic acid(e.g., DNA or RNA) or protein or an organism that would not normally befound in nature and as such was created by human intervention. As usedherein, a “recombinant nucleic acid molecule” (and similar terms) is anucleic acid molecule comprising a combination of polynucleotides thatwould not naturally occur together and is the result of humanintervention, e.g., a nucleic acid molecule that is comprised of acombination of at least two polynucleotides heterologous to each other,or a nucleic acid molecule that is artificially synthesized andcomprises a polynucleotide that deviates from the polynucleotide thatwould normally exist in nature, or a nucleic acid molecule thatcomprises a transgene artificially incorporated into a host cell'sgenomic DNA and the associated flanking DNA of the host cell's genome.An example of a recombinant nucleic acid molecule is a DNA moleculeresulting from the insertion of a transgene into a plant's genomic DNA,which may ultimately result in the expression of a recombinant RNA orprotein molecule in that organism. In embodiments, a “recombinant”protein is a protein that does not normally exist in nature or ispresent in a non-naturally occurring context, and is expressed from arecombinant nucleic acid molecule. As used herein, a “recombinant plant”is a plant that would not normally exist in nature, is the result ofhuman intervention, and contains a recombinant polynucleotide (e.g., atransgene or heterologous nucleic acid molecule incorporated into itsgenome). As a result of such genomic alteration, the recombinant plantis distinctly different from the related wild-type plant.

The terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and“suppress” (and grammatical variations thereof) and similar terms, asused herein, refer to a decrease in the relevant parameter. Inembodiments, the terms “reduce,” “reduced,” “reducing,” “reduction,”“diminish,” and “suppress” (and grammatical variations thereof) andsimilar terms mean a decrease of at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore as compared with a suitable control.

The terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and“suppress” (and grammatical variations thereof) and similar terms, asused herein with reference to control of a plant pest indicate adecrease in the survival, growth and/or reproduction of a plant pest,e.g., by contacting a plant with a polypeptide of the invention (suchas, for example, by transgenic expression or by topical applicationmethods). This decrease in survival, growth and/or reproduction can bein reference to the level observed in the absence of the polypeptide ofthe invention (e.g., a plant that is not transgenically expressing thepolypeptide or is not topically treated with the polypeptide). Thus, inembodiments, the terms “reduce,” “reduced,” “reducing,” “reduction,”“diminish,” and “suppress” (and grammatical variations thereof) andsimilar terms mean a decrease of at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore as compared with a plant that is not contacted with a polypeptideof the invention (e.g., a plant that is not transgenically expressingthe polypeptide or is not topically treated with the polypeptide). Inrepresentative embodiments, the reduction results in no or essentiallyno (i.e., an insignificant amount, e.g., less than about 10%, less thanabout 5% or even less than about 1%) detectable survival, growth and/orreproduction of the plant pest.

A “regulatory element” refers to a nucleotide sequence involved incontrolling the expression of a polynucleotide. Examples of regulatoryelements include promoters, termination signals, and nucleotidesequences that facilitate proper translation of a polynucleotide.

As used herein, “selectable marker” means a nucleotide sequence thatwhen expressed imparts a distinct phenotype to the plant, plant partand/or plant cell expressing the marker and thus allows such transformedplants, plant parts and/or plant cells to be distinguished from thosethat do not have the marker. Such a nucleotide sequence may encodeeither a selectable or screenable marker, depending on whether themarker confers a trait that can be selected for by chemical means, suchas by using a selective agent (e.g., an antibiotic, herbicide, or thelike), or on whether the marker is simply a trait that one can identifythrough observation or testing, such as by screening (e.g., the R-locustrait).

As used herein, “specific activity” refers to the amount of proteinrequired to have an insecticidal effect. Therefore, when a first proteinhas a higher specific activity than a second protein means that it takesa lesser amount of the first protein compared the second protein to havean insecticidal effect on the same percentage of insects.

The phrase “substantially identical,” in the context of two nucleicacids or two amino acid sequences, refers to two or more sequences orsubsequences that have at least about 50% nucleotide or amino acidresidue identity when compared and aligned for maximum correspondence asmeasured using a sequence comparison algorithm or by visual inspection.In certain embodiments, substantially identical sequences have at leastabout 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleotideor amino acid residue identity. In certain embodiments, substantialidentity exists over a region of the sequences that is at least about 50amino acid residues, 100 amino acid residues, 150 amino acid residues,200 amino acid residues, 250 amino acid residues, 300 amino acidresidues, 350 amino acid residues, 400 amino acid residues, 450 aminoacid residues, 500 amino acid residues, 525 amino acid residues, 526,amino acid residues 527 amino acid residues, 528 amino acid residues,529 amino acid residues, 530 amino acid residues, 531 amino acidresidues, 532 amino acid residues, 533 amino acid residues, 534 aminoacid residues, 535 amino acid residues, 536 amino acid residues or morewith respect to the protein sequence or the nucleotide sequence encodingthe same. In further embodiments, the sequences are substantiallyidentical when they are identical over the entire length of the codingregions.

“Identity” or “percent identity” refers to the degree of identitybetween two nucleic acid or amino acid sequences. For sequencecomparison, typically one sequence acts as a reference sequence to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch,J. Mol. Biol. 48: 443 (1970), by the search for similarity method ofPearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (on the world wide web atncbi.nlm.nih.gov/). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when the cumulative alignment scorefalls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see, Henikoff & Henikoff, Proc. Natl. Acad.Sci. USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

Another widely used and accepted computer program for performingsequence alignments is CLUSTALW v1.6 (Thompson, et al. Nuc. Acids Res.,22: 4673-4680, 1994). The number of matching bases or amino acids isdivided by the total number of bases or amino acids, and multiplied by100 to obtain a percent identity. For example, if two 580 base pairsequences had 145 matched bases, they would be 25 percent identical. Ifthe two compared sequences are of different lengths, the number ofmatches is divided by the shorter of the two lengths. For example, ifthere were 100 matched amino acids between a 200 and a 400 amino acidproteins, they are 50 percent identical with respect to the shortersequence. If the shorter sequence is less than 150 bases or 50 aminoacids in length, the number of matches are divided by 150 (for nucleicacid bases) or 50 (for amino acids), and multiplied by 100 to obtain apercent identity.

Two nucleotide sequences can also be considered to be substantiallyidentical when the two sequences hybridize to each other under stringentconditions. In representative embodiments, two nucleotide sequencesconsidered to be substantially identical hybridize to each other underhighly stringent conditions.

The terms “stringent conditions” or “stringent hybridization conditions”include reference to conditions under which a nucleic acid willselectively hybridize to a target sequence to a detectably greaterdegree than other sequences (e.g., at least 2-fold over a non-targetsequence), and optionally may substantially exclude binding tonon-target sequences. Stringent conditions are sequence-dependent andwill vary under different circumstances. By controlling the stringencyof the hybridization and/or washing conditions, target sequences can beidentified that can be up to 100% complementary to the referencenucleotide sequence. Alternatively, conditions of moderate or even lowstringency can be used to allow some mismatching in sequences so thatlower degrees of sequence similarity are detected. For example, thoseskilled in the art will appreciate that to function as a primer orprobe, a nucleic acid sequence only needs to be sufficientlycomplementary to the target sequence to substantially bind thereto so asto form a stable double-stranded structure under the conditionsemployed. Thus, primers or probes can be used under conditions of high,moderate or even low stringency. Likewise, conditions of low or moderatestringency can be advantageous to detect homolog, ortholog and/orparalog sequences having lower degrees of sequence identity than wouldbe identified under highly stringent conditions.

The terms “complementary” or “complementarity” (and similar terms), asused herein, refer to the natural binding of polynucleotides underpermissive salt and temperature conditions by base-pairing. For example,the sequence “A-G-T” binds to the complementary sequence “T-C-A.”Complementarity between two single-stranded molecules may be partial, inwhich only some of the nucleotides bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between themolecules. As used herein, the term “substantially complementary” (andsimilar terms) means that two nucleic acid sequences are at least about50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or morecomplementary. Alternatively, the term “substantially complementary”(and similar terms) can mean that two nucleic acid sequences canhybridize together under high stringency conditions (as describedherein).

As used herein, “specifically” or “selectively” hybridizing (and similarterms) refers to the binding, duplexing, or hybridizing of a molecule toa particular nucleic acid target sequence under stringent conditionswhen that sequence is present in a complex mixture (e.g., total cellularDNA or RNA) to the substantial exclusion of non-target nucleic acids, oreven with no detectable binding, duplexing or hybridizing to non-targetsequences. Specifically or selectively hybridizing sequences typicallyare at least about 40% complementary and are optionally substantiallycomplementary or even completely complementary (i.e., 100% identical).

For DNA-DNA hybrids, the T_(m) can be approximated from the equation ofMeinkoth and Wahl, Anal. Biochem., 138:267-84 (1984): T_(m)=81.5°C.+16.6 (log M)+0.41 (% GC)−0.61 (% formamide)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % formamide is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desireddegree of identity. For example, if sequences with >90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, highly stringent conditions can utilizea hybridization and/or wash at the thermal melting point (T_(m)) or 1,2, 3 or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9 or 10° C. lower than the thermal melting point (T_(m)); low stringencyconditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15or 20° C. lower than the thermal melting point (T_(m)). If the desireddegree of mismatching results in a T_(m) of less than 45° C. (aqueoussolution) or 32° C. (formamide solution), optionally the SSCconcentration can be increased so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, New York (1993); Current Protocols inMolecular Biology, chapter 2, Ausubel, et al., eds, Greene Publishingand Wiley-Interscience, New York (1995); and Green & Sambrook, In:Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2012).

Typically, stringent conditions are those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at about pH 7.0 to pH 8.3and the temperature is at least about 30° C. for short probes (e.g., 10to 50 nucleotides) and at least about 60° C. for longer probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide orDenhardt's (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serumalbumin in 500 ml of water). Exemplary low stringency conditions includehybridization with a buffer solution of 30% to 35% formamide, 1 M NaCl,1% SDS (sodium dodecyl sulfate) at 37° C. and a wash in 1× to 2×SSC(20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50° C. to 55° C.Exemplary moderate stringency conditions include hybridization in 40% to45% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.5× to 1×SSC at55° C. to 60° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in0.1×SSC at 60° C. to 65° C. A further non-limiting example of highstringency conditions include hybridization in 4×SSC, 5×Denhardt's, 0.1mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C. and awash in 0.1×SSC, 0.1% SDS at 65° C. Another illustration of highstringency hybridization conditions includes hybridization in 7% SDS,0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50°C., alternatively with washing in 1×SSC, 0.1% SDS at 50° C.,alternatively with washing in 0.5×SSC, 0.1% SDS at 50° C., oralternatively with washing in 0.1×SSC, 0.1% SDS at 50° C., or even withwashing in 0.1×SSC, 0.1% SDS at 65° C. Those skilled in the art willappreciate that specificity is typically a function ofpost-hybridization washes, the relevant factors being the ionic strengthand temperature of the final wash solution.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the proteins that theyencode are substantially identical (e.g., due to the degeneracy of thegenetic code).

A further indication that two nucleic acids or proteins aresubstantially identical is that the protein encoded by the first nucleicacid is immunologically cross reactive with the protein encoded by thesecond nucleic acid. Thus, a protein is typically substantiallyidentical to a second protein, for example, where the two proteinsdiffer only by conservative substitutions.

As used herein, if a modified polypeptide or fragment (and the like)“substantially retains” insecticidal activity, it is meant that themodified polypeptide or fragment retains at least about 40%, 50%, 60%,70%, 80%, 85%, 90%, 95% or even 100% of the insecticidal activity of thereference protein against one or more target insects, or has evengreater insecticidal activity.

“Synthetic” refers to a nucleotide sequence comprising bases or astructural feature(s) that is not present in the natural sequence. Forexample, an artificial sequence encoding a protein of the invention thatresembles more closely the G+C content and the normal codon distributionof dicot or monocot plant genes is said to be synthetic.

As used herein, a protein that is “toxic” to an insect pest is anorally-active insect control agent that kills the insect pest, causes areduction in growth and/or reproduction of the insect pest and/or isable to disrupt or deter insect feeding, which may or may not causedeath of the insect. When a protein of the invention is delivered to aninsect or an insect comes into contact with the protein, the result istypically death of the insect, the insect's growth and/or reproductionis slowed and/or the insect reduces or stops feeding upon the sourcethat makes the toxic protein available to the insect.

The terms “toxin fragment” and “toxin portion” are used interchangeablyherein to refer to a fragment or portion of a longer (e.g., full-length)insecticidal protein of the invention, where the “toxin fragment” or“toxin portion” retains insecticidal activity. In embodiments, the“toxin fragment” or “toxin portion” of an insecticidal protein of theinvention is truncated at the N-terminus and/or C-terminus. Inembodiments, the “toxin fragment” or “toxin portion” is truncated at theN-terminus, and optionally comprises at least about 405, 410, 425, 450,475, 500, 510, 520, 525, 530, 531, 532, 533, 534, 535, 536, 537 or 538contiguous amino acids of an insecticidal protein specifically describedherein or an amino acid sequence that is substantially identicalthereto. Thus, in embodiments, a “toxin fragment” or “toxin portion” ofan insecticidal protein is truncated at the N-terminus, for example, anN-terminal truncation of one amino acid or more than one amino acid,e.g., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60 or more amino acids, for example, an N-terminaltruncation of the Axmi205 toxin of SEQ ID NO: 1 incorporating one ormore modifications according to the present invention. In embodiments, a“toxin fragment” or “toxin portion” of an insecticidal protein istruncated at the C-terminus, for example, a C-terminal truncation of oneamino acid or more than one amino acid, e.g., up to 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or moreamino acids. In embodiments, the “toxin fragment” or “toxin portion”comprises the MAC/PF (Membrane Attack Complex/Perforin proteinsuperfamily) domain found in the N-terminal region of the native Axmi205protein of SEQ ID NO: 1 (within the region defined by about amino acids101 to 300 of SEQ ID NO: 1; see GenBank Accession No. AML23188.1) or thecorresponding region of other Axmi205 toxins and/or the Beta-Prismdomain in the C-terminal half of the Axmi205 toxin (e.g., within aboutamino acids 300 to 526 SEQ ID NO: 1 or the corresponding region of otherAxmi205 toxins).

“Transformation” is a process for introducing a heterologous nucleicacid into a host cell or organism. In particular embodiments,“transformation” means the stable integration of a DNA molecule into thegenome of an organism of interest (e.g., a plant cell).

The terms “transformed” and “transgenic” as used herein refer to a hostorganism such as a bacterium or a plant into which a heterologousnucleic acid molecule has been introduced. The nucleic acid molecule canbe stably integrated into the genome of the host or the nucleic acidmolecule can also be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. “Transformed” or“transgenic” cells, tissues, or plants are understood to encompass notonly the end product of a transformation process, but also progenythereof comprising the heterologous nucleic acid molecule. A“non-transformed” or “non-transgenic” host refers to a wild-typeorganism, e.g., a bacterium or plant, which does not contain theheterologous nucleic acid molecule.

The term “vector” refers to a composition for transferring, deliveringor introducing a nucleic acid (or nucleic acids) into a cell. A vectorcomprises a nucleic acid molecule comprising the nucleotide sequence(s)to be transferred, delivered or introduced.

Pesticidal Proteins.

The present invention provides novel pesticidal (e.g., insecticidal)proteins comprising a modified Axmi205 toxin that has enhanced digestion(e.g., cleavage) by a digestive protease (e.g., pepsin) as compared withan Axmi205 toxin that does not comprise the modification (e.g., theAxmi205 protein of SEQ ID NO: 1 and/or an Axmi205 toxin that isidentical to the modified Axmi205 protein of the invention with theexception that it lacks the modifications of the present invention).Modifications within the scope of the present invention include withoutlimitations deletions, substitutions and/or insertions.

The native Axmi205 toxin of SEQ ID NO: 1 has previously been describedin U.S. Pat. No. 8,575,425 B2 and Sampson et al. (Discovery of a novelinsecticidal protein from Chromobacterium piscinae with activity againstWestern Corn Rootworm, Diabrotica virgifera virgifera, J. InvertebratePathology 142: 34-43 (2016)). See also GenBank Accession No. AML23188.1.U.S. Pat. No. 8,575,425 B2 also describes a number of Axmi205 pointmutations and truncations that retain activity against WCRW (see,Examples 7 and 8 of U.S. Pat. No. 8,575,425 B2). Such mutants includethe following mutations in the Axmi205 protein sequence of SEQ ID NO: 1of the present application (SEQ ID NO: 2 of U.S. Pat. No. 8,575,425 B2):S307A, D315A, V317A, S349A, G351A, K353A, V355A, D395A, G399A, W407A,G419A, P355A, P435A, S443A, K465A, V467A, F483A, P487A, S495A, D497A,E499A, K509A and I513A. Also disclosed are Axmi205 proteins havingC-terminal truncations of 10 or 20 amino acids from the C-terminus ofthe Axmi205 of SEQ ID NO: 1 of the present application (SEQ ID NO: 2 ofU.S. Pat. No. 8,575,425 B2).

As used herein, an “Axmi205 toxin” to be modified according to thepresent invention (e.g., to enhance digestion by a digestive proteasesuch as pepsin) comprises, consists essentially of, or consists of theamino acid sequence of SEQ ID NO: 1 or an amino acid sequence that issubstantially identical to SEQ ID NO: 1. Generally, the Axmi205 toxin tobe modified according to the present invention has an undesirabledigestion profile by a mammalian digestive protease (e.g., pepsin),e.g., undigested or partially digested fragments of the Axmi205 toxinabove about 4 kDa remain after about 15, 20, 25, 30, 35, 40, 45, 50, 55,60 or more minutes of digestion with the digestive protease, optionallyunder conditions of approximately 37° C. and approximately pH 1.2, andas a further option with a protease concentration of 10 U per microgramprotein. Thus, the modifications disclosed herein can be incorporatedinto the Axmi205 toxin so as to enhance/improve the digestion by adigestive protease (e.g., protease).

The Axmi205 toxin of SEQ ID NO: 1 was isolated from Chromobacteriumpiscinae. In embodiments, as used herein, an “Axmi205 toxin” to bemodified according to the present invention is from the genusChromobacterium, optionally C. piscinae (either isolated directly fromthe organism, or partly or completely synthesized to replicate anaturally occurring Chromobacterium, optionally C. piscinae, protein).United States patent publication US2014/0223599 (Athenix) describesAxmi279 (SEQ ID NO: 2 of that patent publication), which was alsoisolated from C. piscinae, has 97.9% amino acid identity with Axmi205,and is active in controlling WCRW. US2014/0223599 also describes twoAxmi279 variants from which the C-terminal amino acid and the N-terminal18 or 20 amino acids are truncated (SEQ ID NO: 3 and SEQ ID NO: 4,respectively, of US2014/0223599). The Axmi279 protein and variantsthereof described in US2014/0223599 are also encompassed by the Axmi205toxins according to the present invention.

WO2013/016617 (Athenix) also describes a number of variants of theAxmi205 protein of SEQ ID NO: 1 of the present application: S468L,V467L, V467T, R464N, Q517R and E86T having activity against WCRW.Furthermore, US 2014/0274885 A1 (Pioneer Hi-Bred) also describes a largenumber of Axmi205 variants having insecticidal activity against WCRW.

Thus, in embodiments, as used herein an “Axmi205 toxin” to be modifiedaccording to the present invention comprises, consists essentially of,or consists of a variant Axmi205 protein, for example, the Axmi205toxins described in U.S. Pat. No. 8,575,425 B2 (Athenix), WO2013/016617(Athenix) or in US 2014/0274885 A1 (Pioneer Hi-Bred)).

Accordingly, the term “Axmi205 toxin” as used herein encompasses SEQ IDNO: 1 as well as the Axmi205 variants disclosed in U.S. Pat. No.8,575,425 B2, WO2013/016617 and/or US 2014/0274885 A1, and/or Axmi279and variants thereof disclosed in US2014/0223599.

In embodiments, the modified Axmi205 toxin comprises a deletion(including a truncation), substitution and/or insertion of one or moreamino acids as compared with the Axmi205 toxin of SEQ ID NO: 1 or asubstantially identical protein, wherein the deletion, substitutionand/or insertion results in enhanced digestion of the modified Axmi205toxin by pepsin and/or other mammalian digestive proteases (e.g., humandigestive proteases) such as trypsin and/or chymotrypsin as comparedwith an Axmi205 toxin that does not comprise the deletion, substitutionand/or insertion (e.g., SEQ ID NO: 1 and/or an Axmi205 toxin that isidentical to the modified Axmi205 protein of the invention with theexception that it lacks the modifications of the present invention).

In embodiments, the modified Axmi205 toxins of the inventionsubstantially retain the insecticidal activity of the parent molecule(e.g., SEQ ID NO:1) against one or more target pests, e.g., acoleopteran pest such as WCRW.

In embodiments, the insecticidal proteins of the invention can providenew modes of action against one or more target insect pests. Forexample, an insecticidal protein of the invention can have insecticidalactivity against an insect pest or colony that is generally resistant tothe insecticidal activity of another insect control agent, e.g., aninsecticidal protein or an insecticidal dsRNA. To illustrate, theinsecticidal protein of the invention may have insecticidal activityagainst a corn rootworm (e.g., WCRW) pest or colony that is resistant toa mCry3A protein (e.g., in corn event MIR604), an eCry3.1Ab protein(e.g., in corn event 5307), a Cry3Bb1 protein (e.g., in corn eventMON88017), a Cry34/35Ab1 binary protein (e.g., in corn event DAS-59122)and/or a RNAi trait, such as DvSnf7 dsRNA (e.g., in corn eventMON87411).

In embodiments, the modified Axmi205 proteins of the invention haveenhanced digestion by a mammalian digestive protease (e.g., pepsin) ascompared with a suitable control (e.g., SEQ ID NO: 1 and/or the parentalmolecule not containing a modification of the invention) when testedunder the same conditions (e.g., enzyme concentration, proteinconcentration, pH, temperature and/or time). Methods for assessingprotein digestion by pepsin and other digestive proteases are well-knownin the art, for example, the Simulated Gastric Fluid (SGF) assaydescribed in Examples 3 and 5. For example, digestion with pepsin can becarried out at approximately 37° C. and approximately pH 1.2, optionallywith an enzyme concentration of approximately 10 Units (U) pepsin permicrogram of protein.

In representative embodiments, no detectable fragments (e.g.,immunoreactive fragments) of the modified Axmi205 toxin of the inventionremain above approximately 4 kDa after about 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes of digestionwith the digestive protease (e.g., pepsin), optionally under theconditions described in the preceding paragraph. Methods of detectingundigested or partially digested fragments of the modified Axmi205protein are known in the art (e.g., SDS-PAGE), and immunoreactivefragments can be detected with a suitable antibody (e.g., directedagainst the Axmi205 toxin of SEQ ID NO: 1), for example, as described inthe working Examples.

In embodiments, the modified Axmi205 toxin comprises the MAC/PF(Membrane Attack Complex/Perforin protein superfamily) domain found inthe N-terminal region of the native Axmi205 protein of SEQ ID NO: 1(within the region defined by about amino acids 101 to 300 of SEQ ID NO:1; see GenBank: AML23188.1) and/or the Beta-Prism domain in theC-terminal half of the Axmi205 toxin (e.g., within about amino acids 300to 526 SEQ ID NO: 1 or the corresponding region of other Axmi205toxins).

The modification(s) of the invention can be made in any portion(s) ofthe parental Axmi205 toxin that results in an enhanced digestion by amammalian digestive protease (e.g., pepsin), optionally with substantialretention of the insecticidal activity of the parental Axmi205 toxin. Inembodiments, the Axmi205 toxin is modified by deletion, substitutionand/or insertion of one or more amino acids in a portion of the Axmi205toxin of SEQ ID NO: 1 from about amino acid 400 or 402 to about aminoacid 497, 500, or 536, or the corresponding portion of another Axmi205toxin. In embodiments, the deletion, substitution and/or insertion ofone or more amino acids is in a portion of the Axmi205 toxin of SEQ IDNO: 1 from about amino acid 402 to about amino acid 497, or thecorresponding portion of another Axmi205 toxin.

In representative embodiments, the modification can comprisesubstitution and/or insertion of one or more of any naturally-occurringand/or non-naturally occurring amino acid. In embodiments, themodification comprises an insertion and/or substitution of one or moreof alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine and/or valine. In embodiments, the insertion and/orsubstitution is not an alanine.

In embodiments, the Axmi205 toxin is modified by substitution and/orinsertion of (a) one or more amino acids with an aliphatic hydrophobicside chain (e.g., alanine, isoleucine, methionine and/or valine; inembodiments, the amino acid is not an alanine); (b) one or more aminoacids with an aromatic hydrophobic side chain (e.g., phenylalanine,tryptophan and/or tyrosine); (c) one or more amino acids with a polarneutral side chain (e.g., asparagine, cysteine, glutamine, serine and/orthreonine); (d) one or more amino acids with an acidic side chain (e.g.,aspartic acid and/or glutamic acid); one or more amino acids with abasic side chain (e.g., arginine, histidine and/or lysine); (e) one ormore glycine residues; (f) one or more proline residues; or (g) anycombination of (a) to (f).

In representative embodiments, the deletion, substitution and/orinsertion of one or more amino acids creates a new cleavage site for amammalian digestive protease (e.g., pepsin) that did not exist in theparent Axmi205 toxin, for example, a non-naturally occurring pepsincleavage site incorporated into the Axmi205 toxin of SEQ ID NO: 1. Toillustrate, as is known in the art, pepsin preferentially cleavesbetween two hydrophobic amino acids (e.g., alanine, isoleucine, valine,phenylalanine, tryptophan and/or tyrosine). Thus, in embodiments, themodification comprises the insertion or substitution of a hydrophobicamino acid (with an aliphatic and/or aromatic side chain) adjacent to anexisting hydrophobic amino acid to create a new pepsin cleavage site. Inembodiments, the modification comprises insertion or substitution of twoamino acids to create two adjacent hydrophobic amino acids. Inembodiments, one amino acid is substituted and one amino acid insertedto create two adjacent hydrophobic amino acids. In representativeembodiments, one or more amino acids with an aliphatic hydrophobic sidechain and/or an aromatic hydrophobic side chain are substituted and/orinserted. In further representative embodiments, a deletion of one ormore (e.g., 2, 3, 4 or 5) amino acids brings two hydrophobic amino acidsinto adjacent positions so as to create a protease cleavage site.

Without being bound by any theory of the invention, in embodiments, themodification to the Axmi205 toxin opens up the secondary and/or tertiarystructure of the protein thereby providing better access to digestiveproteases. In embodiments, the modification to the Axmi205 toxincomprises a deletion or substitution of one or more cysteine residues(e.g., 1, 2, 3, 4, or 5 cysteine residues) by another amino acidresidue, optionally to reduce potential disulfide bond formation (e.g.,by reducing the total number of cysteine residues in the protein).

In embodiments, the modified Axmi205 toxin comprises: (a) an amino acidsubstitution at amino acid K402 in the polypeptide of SEQ ID NO:1 or thecorresponding lysine residue in another Axmi205 toxin; (b) amino acidsubstitutions at amino acids K402 and Y404 in the polypeptide of SEQ IDNO:1 or the corresponding lysine and tyrosine residues in anotherAxmi205 toxin; (c) an amino acid substitution at amino acid C482 in thepolypeptide of SEQ ID NO:1 or the corresponding cysteine residue inanother Axmi205 toxin; (d) an amino acid substitution at amino acid C507in the polypeptide of SEQ ID NO:1 or the corresponding cysteine residuein another Axmi205 toxin; (e) amino acid substitutions at amino acidsM422 and M423 in the polypeptide of SEQ ID NO:1 or the correspondingmethionine residues in another Axmi205 toxin; (f) an amino acidinsertion between amino acids A475 and G476 in the polypeptide of SEQ IDNO:1 or the corresponding alanine and glycine residues in anotherAxmi205 toxin; (g) an amino acid insertion between amino acids G496 andD497 in the polypeptide of SEQ ID NO:1 or the corresponding glycine andaspartic acid residues in another Axmi205 toxin; (h) an amino acidinsertion between amino acids Q471 and P472 in the polypeptide of SEQ IDNO:1 or the corresponding glycine and aspartic acid residues in anotherAxmi205 toxin; (i) an amino acid insertion between amino acids A479 andS480 in the polypeptide of SEQ ID NO:1 or the corresponding glycine andaspartic acid residues in another Axmi205 toxin; (j) an amino acidinsertion between amino acids Y489 and N490 in the polypeptide of SEQ IDNO:1 or the corresponding glycine and aspartic acid residues in anotherAxmi205 toxin; or (k) any combination of (a) to (j) above).

In embodiments, the modified Axmi205 toxin comprises: a substitution ofan amino acid with an aliphatic hydrophobic side chain, an amino acidwith an aromatic hydrophobic side chain, an amino acid with a polarneutral side chain, an amino acid with an acidic side chain, or an aminoacid with a basic side chain at amino acid K402 in the polypeptide ofSEQ ID NO:1 or the corresponding lysine in another Axmi205 toxin;

-   -   a) amino acid substitutions of (i) an amino acid with an        aliphatic hydrophobic side chain, an amino acid with an aromatic        hydrophobic side chain, an amino acid with a polar neutral side        chain, an amino acid with an acidic side chain, an amino acid        with a basic side chain, a glycine or a proline at amino acid        K402 in the polypeptide of SEQ ID NO:1 or the corresponding        lysine residue in another Axmi205 toxin; and (ii) an amino acid        with an aliphatic hydrophobic side chain, an amino acid with an        aromatic hydrophobic side chain, an amino acid with a polar        neutral side chain, an amino acid with an acidic side chain, an        amino acid with a basic side chain, a glycine or a proline at        amino acid Y404 in the polypeptide of SEQ ID NO:1 or the        corresponding tyrosine residue in another Axmi205 toxin;    -   b) a substitution of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, an amino acid with a basic side        chain, a glycine or a proline at amino acid C482 in the        polypeptide of SEQ ID NO:1 or the corresponding cysteine residue        in another Axmi205 toxin;    -   c) a substitution of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, an amino acid with a basic side        chain, a glycine or a proline at amino acid C507 in the        polypeptide of SEQ ID NO:1 or the corresponding cysteine residue        in another Axmi205 toxin;    -   d) amino acid substitutions of (i) an amino acid with an        aliphatic hydrophobic side chain, an amino acid with an aromatic        hydrophobic side chain, an amino acid with a polar neutral side        chain, an amino acid with an acidic side chain, an amino acid        with a basic side chain, a glycine or a proline at amino acid        M422 in the polypeptide of SEQ ID NO:1 or the corresponding        methionine residue in another Axmi205 toxin; and (ii) an amino        acid with an aliphatic hydrophobic side chain, an amino acid        with an aromatic hydrophobic side chain, an amino acid with a        polar neutral side chain, an amino acid with an acidic side        chain, an amino acid with a basic side chain, a glycine or a        proline at amino acid M423 in the polypeptide of SEQ ID NO:1 or        the corresponding methionine residue in another Axmi205 toxin;    -   e) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, an amino acid with a basic side        chain, a glycine or a proline between amino acids A475 and G476        in the polypeptide of SEQ ID NO:1 or the corresponding alanine        and glycine residues in another Axmi205 toxin;    -   f) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, an amino acid with a basic side        chain, a glycine or a proline between amino acids G496 and D497        in the polypeptide of SEQ ID NO:1 or the corresponding glycine        and aspartic acid residues in another Axmi205 toxin;    -   g) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain between amino acids Q471 and P472 in the polypeptide        of SEQ ID NO:1 or the corresponding alanine and glycine residues        in another Axmi205 toxin;    -   h) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain between amino acids A479 and S480 in the polypeptide        of SEQ ID NO:1 or the corresponding alanine and glycine residues        in another Axmi205 toxin;    -   i) an insertion of an amino acid with an aliphatic hydrophobic        side chain, an amino acid with an aromatic hydrophobic side        chain, an amino acid with a polar neutral side chain, an amino        acid with an acidic side chain, or an amino acid with a basic        side chain between amino acids Y489 and N490 in the polypeptide        of SEQ ID NO:1 or the corresponding alanine and glycine residues        in another Axmi205 toxin; or    -   j) any combination of (a) to (i).

Amino acids with an aliphatic hydrophobic side chain, an aromatichydrophobic side chain, a polar neutral side chain, an acidic sidechain, or a basic side chain are as described elsewhere herein.

In exemplary embodiments, the modified Axmi205 toxin comprises:

-   -   a) an amino acid substitution of K402F, K402N or K402D in the        polypeptide of SEQ ID NO:1 or the corresponding lysine in        another Axmi205 toxin;    -   b) amino acid substitutions of (i) K402L and Y404F, or (ii)        K402D and Y404L in the polypeptide of SEQ ID NO:1 or the        corresponding lysine and tyrosine residues in another Axmi205        toxin;    -   c) an amino acid substitution of C482S, C482D or C482F in the        polypeptide of SEQ ID NO:1 or the corresponding cysteine residue        in another Axmi205 toxin;    -   d) an amino acid substitution of C507S, C507L, C507A, C507F,        C507D or C507R in the polypeptide of SEQ ID NO:1 or the        corresponding cysteine residue in another Axmi205 toxin;    -   e) amino acid substitutions of (i) M422S and M423L, (ii) M422T        and M423F, (iii) M422K and M423F, (iv) M422D and M423E, (v)        M422K and M423R, or (vi) M422S and M423E, or the corresponding        methionine residues in another Axmi205 toxin;    -   f) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids A475 and G476 in the polypeptide of        SEQ ID NO:1 or the corresponding alanine and glycine residues in        another Axmi205 toxin;    -   g) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids G496 and D497 in the polypeptide of        SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin;    -   h) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids Q471 and P472 in the polypeptide of        SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin;    -   i) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids A479 and S480 in the polypeptide of        SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin;    -   j) an insertion of a leucine, phenylalanine, aspartic acid or        arginine between amino acids Y489 and N490 in the polypeptide of        SEQ ID NO:1 or the corresponding glycine and aspartic acid        residues in another Axmi205 toxin; or    -   k) any combination of (a) to (j).

In particular embodiments, a insecticidal protein of the inventioncomprises, consists essentially of, or consists of (a) the amino acidsequence of any one of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQID NO: 43, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 69, or SEQ ID NO:73, or a toxin fragment thereof; or (b) an amino acid sequence that issubstantially identical to the amino acid sequence of (a).

Those skilled in the art will appreciate that the insecticidal proteinsof the invention can further comprise other functional domains and/orpeptide tags, for example a peptide tag on the N-terminus and/orC-terminus. To illustrate, it may be useful to express the insecticidalprotein with a peptide tag that can be recognized by a commerciallyavailable antibody (e.g., a FLAG motif) or with a peptide tag thatfacilitates purification (e.g., by addition of a poly-His tag) and/ordetection. Alternatively, an epitope can be introduced into the proteinto facilitate the generation of antibodies that specifically recognizethe modified protein to distinguish the modified protein from theunmodified chimera and/or a parent protein(s). For example, one or moreamino acids can be substituted into an antigenic loop to create a newepitope. In other embodiments, the protein can be modified to enhanceits stability, for example, by fusing a maltose binding protein (MBP) orglutathione-S-transferase to the polypeptide. As another alternative,the insecticidal protein can be a fusion protein comprising a reportermolecule. Further, sub-cellular targeting peptides can be incorporatedinto the protein, such as a KDEL sequence tag that targets to theendoplasmic reticulum.

As discussed above, the invention encompasses polypeptides having aminoacid sequences that are substantially identical to those specificallydisclosed herein, and toxin fragments thereof. It will be understoodthat the insecticidal proteins specifically disclosed herein willtypically tolerate modifications in the amino acid sequence andsubstantially retain biological activity (e.g., insecticidal activity).Such modifications include insertions, deletions (including truncationsat either terminus), and substitutions of one or more amino acids,including up to about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 100, about 105,about 110, about 115, about 120, about 125, about 130, about 135, about140, about 145, about 150, about 155, or more amino acid substitutions,deletions and/or insertions.

In embodiments, the polypeptide of the invention comprises amodification as disclosed in WO2013/016617 (Athenix) and/or US2014/0274885 A1 (Pioneer Hi-Bred).

To identify substantially identical polypeptides to the insecticidalproteins specifically disclosed herein, amino acid substitutions may bebased on any characteristic known in the art, including the relativesimilarity or differences of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. For example, in identifying amino acid sequences encodinginsecticidal polypeptides other than those specifically disclosedherein, the hydropathic index of amino acids may be considered. Theimportance of the hydropathic amino acid index in conferring interactivebiologic function on a protein is generally understood in the art (see,Kyte and Doolittle, (1982) J. Mol. Biol. 157:105; incorporated herein byreference in its entirety). It is accepted that the relative hydropathiccharacter of the amino acid contributes to the secondary structure ofthe resultant protein, which in turn defines the interaction of theprotein with other molecules, for example, enzymes, substrates,receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle, Id.),these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

Accordingly, the hydropathic index of the amino acid (or amino acidsequence) may be considered when modifying the polypeptides specificallydisclosed herein.

It is also understood in the art that the substitution of amino acidscan be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (.+−.3.0); aspartate (+3.0.+−0.1); glutamate (+3.0.+−0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5+1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Thus, the hydrophilicity of the amino acid (or amino acid sequence) maybe considered when identifying additional insecticidal polypeptidesbeyond those specifically disclosed herein.

The insecticidal proteins of the invention, including modifications andtoxin fragments of the polypeptides specifically disclosed herein, canbe made by any suitable method known in the art, generally by modifyingthe coding nucleic acid sequences. Methods of manipulating and modifyingnucleic acids to achieve a desired modification are well-known in theart. In addition, gene editing techniques can also be used produce aninsecticidal protein of the invention or to make further modificationsthereto.

As another approach, the polypeptide to be modified can be expressed ina host cell that exhibits a high rate of base mis-incorporation duringDNA replication, such as XL-1 Red (Stratagene, La Jolla, Calif.). Afterpropagation in such strains, one can isolate the DNA (for example, bypreparing plasmid DNA or by PCR amplification and cloning of theresulting PCR fragment into a vector), culture the protein mutations ina non-mutagenic strain, and identify mutated genes with insecticidalactivity, for example, by performing an assay to test for insecticidalactivity. In exemplary methods, the protein is mixed and used in feedingassays. See, for example, Marrone et al. (1985) J. of EconomicEntomology 78:290-293. Such assays can include contacting plants withone or more pests and determining the plant's ability to survive orcause the death of the pests. Examples of mutations that result inincreased toxicity are found in Schnepf et al. (1998) Microbiol. Mol.Biol. Rev. 62:775-806.

In embodiments, the insecticidal protein (including substantiallysimilar polypeptides and toxin fragments) of the invention is isolated.In embodiments, the insecticidal protein (including substantiallysimilar polypeptides and toxin fragments) of the invention is arecombinant protein.

Variants of the insecticidal proteins of the invention can be generatedby any method known in the art including genome editing technologies.For example, after a heterologous polynucleotide sequence encoding ininsecticidal protein encompassed by the invention is introduced into aplant the introduced polynucleotide is stably integrated into the genomeof the now transgenic plant. Thus, according to the invention, theencoded insecticidal protein can be further modified in situ by targetedDNA editing using various genome editing techniques such as zinc fingernucleases (ZNFs), transcription activator-like effector nucleases(TALENS), meganucleases and Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) (U.S. Pat. No. 8,697,359; Ran et al.). TheCRISPR system can be used to introduce specific nucleotide modificationsat the target sequence. Originally discovered in bacteria, where severaldifferent CRISPR cascades function as innate immune systems and naturaldefense mechanisms, the engineered CRISPR-Cas9 system can be programmedto target specific stretches of genetic code and to make cuts at preciselocations. Over the past few years, those capabilities have beenharnessed and used as genome editing tools, enabling researchers topermanently modify genes in plant cells.

The insecticidal proteins of the invention have activity against one ormore insect pests. In embodiments, the insecticidal proteins of theinvention have activity against a coleopteran, lepidopteran, dipteran,hemipteran, orthopteran and/or thysanopteran insect pest. Inembodiments, the insecticidal protein is active against a coleopteranpest.

Insects in the order Coleoptera include but are not limited to anycoleopteran insect now known or later identified including those insuborders Archostemata, Myxophaga, Adephaga and Polyphaga, and anycombination thereof.

In one aspect of this embodiment, the insecticidal proteins of theinvention are active against Diabrotica spp. Diabrotica is a genus ofbeetles of the order Coleoptera commonly referred to as “corn rootworms”or “cucumber beetles.” Exemplary Diabrotica species include withoutlimitation Diabrotica barberi (northern corn rootworm), D. virgiferavirgifera (western corn rootworm), D. undecimpunctata howardii (southerncorn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctataundecimpunctata (western spotted cucumber beetle), D. significata(3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), D.virgifera zeae (Mexican corn rootworm), D. beniensis, D. cristata, D.curviplustalata, D. dissimilis, D. elegantula, D. emorsitans, D.graminea, D. hispanloe, D. lemniscata, D. linsleyi, D. milleri, D.nummularis, D. occlusal, D. porrecea, D. scutellata, D. tibialis, D.trifasciata and D. viridula; and any combination thereof.

Other nonlimiting examples of Coleopteran insect pests according to thepresent invention include Leptinotarsa spp. such as L. decemlineata(Colorado potato beetle); Chrysomela spp. such as C. scripta (cottonwoodleaf beetle); Hypothenemus spp. such as H. hampei (coffee berry borer);Sitophilus spp. such as S. zeamais (maize weevil); Epitrix spp. such asE. hirtipennis (tobacco flea beetle) and E. cucumeris (potato fleabeetle); Phyllotreta spp. such as P. cruciferae (crucifer flea beetle)and P. pusilla (western black flea beetle); Anthonomus spp. such as A.grandis (boll weevil) and A. eugenii (pepper weevil); Hemicrepidus spp.such as H. memnonius (wireworms); Melanotus spp. such as M. communis(wireworm); Ceutorhychus spp. such as C. assimilis (cabbage seedpodweevil); Phyllotreta spp. such as P. cruciferae (crucifer flea beetle);Aeolus spp. such as A. mellillus (wireworm); Aeolus spp. such as A.mancus (wheat wireworm); Horistonotus spp. such as H. uhlerii (sandwireworm); Sphenophorus spp. such as S. maidis (maize billbug), S. zeae(timothy billbug), S. parvulus (bluegrass billbug), and S. callosus(southern corn billbug); Phyllophaga spp. (White grubs); Chaetocnemaspp. such as C. pulicaria (corn flea beetle); Popillia spp. such as P.japonica (Japanese beetle); Epilachna spp. such as E. varivestis(Mexican bean beetle); Cerotoma spp. such as C. trifurcate (Bean leafbeetle); Epicauta spp. such as E. pestifera and E. lemniscata (Blisterbeetles); and any combination of the foregoing.

In embodiments, the insecticidal protein has activity against one ormore of the following non-limiting examples of a lepidopteran pest:Ostrinia spp. such as O. nubilalis (European corn borer) and/or O.fumacalis (Asian corn borer); Plutella spp. such as P. xylostella(diamondback moth); Spodoptera spp. such as S. frugiperda (fallarmyworm), S. littoralis (Egyptian cotton leafworm), S. ornithogalli(yellowstriped armyworm), S. praefica (western yellowstriped armyworm),S. eridania (southern armyworm) and/or S. exigua (beet armyworm);Agrotis spp. such as A. ipsilon (black cutworm), A. segetum (commoncutworm), A. gladiaria (claybacked cutworm), and/or A. orthogonia (palewestern cutworm); Striacosta spp. such as S. albicosta (western beancutworm); Helicoverpa spp. such as H. zea (corn earworm), H. punctigera(native budworm), and/or H. armigera (cotton bollworm); Heliothis spp.such as H. virescens (tobacco budworm); Diatraea spp. such as D.grandiosella (southwestern corn borer) and/or D. saccharalis (sugarcaneborer); Trichoplusia spp. such as T. ni (cabbage looper); Sesamia spp.such as S. nonagroides (Mediterranean corn borer) and/or S. calamistis(pink stem borer); Pectinophora spp. such as P. gossypiella (pinkbollworm); Cochylis spp. such as C. hospes (banded sunflower moth);Manduca spp. such as M. sexta (tobacco hornworm) and/or M.quinquemaculata (tomato hornworm); Elasmopalpus spp. such as E.lignosellus (lesser cornstalk borer); Pseudoplusia spp. such as P.includens (soybean looper); Anticarsia spp. such as A. gemmatalis(velvetbean caterpillar); Plathypena spp. such as P. scabra (greencloverworm); Pieris spp. such as P. brassicae (cabbage butterfly),Papaipema spp. such as P. nebris (stalk borer); Pseudaletia spp. such asP. unipuncta (common armyworm); Peridroma spp. such as P. saucia(variegated cutworm); Keiferia spp. such as K. lycopersicella (tomatopinworm); Artogeia spp. such as A. rapae (imported cabbageworm);Phthorimaea spp. such as P. operculella (potato tuberworm); Chrysodeixisspp. such as C. includes (soybean looper); Feltia spp. such as F. ducens(dingy cutworm); Chilo spp. such as C. suppressalis (striped stemborer), Cnaphalocrocis spp. such as C. medinalis (rice leaffolder), orany combination of the foregoing.

Insects in the order Hemiptera include Lygus spp. stink bugs (includingNezara spp., Halyomorpha spp, Brochymena spp., and Euschistus spp.),aphids (including Aphis spp. and Nasonovia spp.), and other piercing andsucking insects.

Insects in the order Diptera include but are not limited to any dipteraninsect now known or later identified including but not limited toLiriomyza spp. such as L. trifolii (leafminer) and L. sativae (vegetableleafminer); Scrobipalpula spp. such as S. absoluta (tomato leafminer);Delia spp. such as D. platura (seedcorn maggot), D. brassicae (cabbagemaggot) and D. radicum (cabbage root fly); Psilia spp. such as P. rosae(carrot rust fly); Tetanops spp. such as T. myopaeformis (sugarbeet rootmaggot); and any combination of the foregoing.

Insects in the order Orthoptera include but are not limited to anyorthopteran insect now known or later identified including but notlimited to Melanoplus spp. such as M. differentialis (Differentialgrasshopper), M. femurrubrum (Redlegged grasshopper), M. bivittatus(Twostriped grasshopper); and any combination thereof.

Insects in the order Thysanoptera include but are not limited to anythysanopteran insect now known or later identified including but notlimited to Frankliniella spp. such as F. occidentalis (western flowerthrips) and F. fusca (tobacco thrips); and Thrips spp. such as T. tabaci(onion thrips), T. palmi (melon thrips); and any combination of theforegoing.

In embodiments, the insecticidal proteins of the invention are activeagainst nematodes. The term “nematode” as used herein encompasses anyorganism that is now known or later identified that is classified in theanimal kingdom, phylum Nematoda, including without limitation nematodeswithin class Adenophorea (including for example, orders Enoplida,Isolaimida, Mononchida, Dorylaimida, Trichocephalida, Mermithida,Muspiceida, Araeolaimida, Chromadorida, Desmoscolecida, Desmodorida andMonhysterida) and/or class Secernentea (including, for example, ordersRhabdita, Strongylida, Ascaridida, Spirurida, Camallanida,Diplogasterida, Tylenchida and Aphelenchida).

Nematodes include but are not limited to parasitic nematodes such asroot-knot nematodes, cyst nematodes and/or lesion nematodes. Exemplarygenera of nematodes according to the present invention include but arenot limited to, Meloidogyne (root-knot nematodes), Heterodera (cystnematodes), Globodera (cyst nematodes), Radopholus (burrowingnematodes), Rotylenchulus (reniform nematodes), Pratylenchus (lesionnematodes), Aphelenchoides (foliar nematodes), Helicotylenchus (spiralnematodes), Hoplolaimus (lance nematodes), Paratrichodorus (stubby-rootnematodes), Longidorus, Nacobbus (false root-knot nematodes),Subanguina, Belonlaimus (sting nematodes), Criconemella, Criconemoides(ring nematodes), Ditylenchus, Dolichodorus, Hemicriconemoides,Hemicycliophora, Hirschmaniella, Hypsoperine, Macroposthonia, Melinius,Punctodera, Quinisulcius, Scutellonema, Xiphinema (dagger nematodes),Tylenchorhynchus (stunt nematodes), Tylenchulus, Bursaphelenchus (roundworms), and any combination thereof.

Exemplary plant parasitic nematodes according to the present inventioninclude, but are not limited to, Belonolaimus gracilis, Belonolaimuslongicaudatus, Bursaphelenchus xylophilus (pine wood nematode),Criconemoides ornata, Ditylenchus destructor (potato rot nematode),Ditylenchus dipsaci (stem and bulb nematode), Globodera pallida (potatocyst nematode), Globodera rostochiensis (golden nematode), Heteroderaglycines (soybean cyst nematode), Heterodera schachtii (sugar beet cystnematode); Heterodera zeae (corn cyst nematode), Heterodera avenae(cereal cyst nematode), Heterodera carotae, Heterodera trifolii,Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimus magnistylus,Longidorus breviannulatus, Meloidogyne arenaria, Meloidogyne chitwoodi,Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica,Mesocriconema xenoplax, Nacobbus aberrans, Naccobus dorsalis,Paratrichodorus christiei, Paratrichodorus minor, Pratylenchusbrachyurus, Pratylenchus crenatus, Pratylenchus hexincisus, Pratylenchusneglectus, Pratylenchus penetrans, Pratylenchus projectus, Pratylenchusscribneri, Pratylenchus tenuicaudatus, Pratylenchus thornei,Pratylenchus zeae, Punctodera chaccoensis, Quinisulcius acutus,Radopholus similis, Rotylenchulus reniformis, Tylenchorhynchus dubius,Tylenchulus semipenetrans (citrus nematode), Siphinema americanum, X.Mediterraneum, and any combination of the foregoing.

The invention also encompasses antibodies that specifically bind to theinsecticidal proteins of the invention. The antibody can optionally be amonoclonal antibody or a polyclonal antisera. In embodiments, theantibody is selective for the modified Axmi205 protein and does not bindto the unmodified Axmi205 toxin, e.g., the native Axmi205 protein of SEQID NO: 1, and can be used to distinguish the modified protein from theunmodified Axmi205 protein. Such antibodies may be produced usingstandard immunological techniques for production of polyclonal antiseraand, if desired, immortalizing the antibody-producing cells of theimmunized host for sources of monoclonal antibody production. Techniquesfor producing antibodies to any substance of interest are well known,e.g., as described in Harlow and Lane (1988. Antibodies a laboratorymanual. pp. 726. Cold Spring Harbor Laboratory) and as in Goding(Monoclonal Antibodies: Principles & practice.1986. Academic Press,Inc., Orlando, Fla.). The present invention also encompasses aninsecticidal protein that cross-reacts with an antibody, particularly amonoclonal antibody, raised against one or more of the insecticidalproteins of the present invention.

The antibodies according to the invention are useful, e.g., inimmunoassays for determining the amount or presence of an insecticidalprotein of the invention or an antigenically related polypeptide, e.g.,in a biological sample. Such assays are also useful inquality-controlled production of compositions containing one or more ofthe insecticidal proteins of the invention or an antigenically relatedpolypeptide. In addition, the antibodies can be used to assess theefficacy of recombinant production of one or more of the insecticidalproteins of the invention or an antigenically related polypeptide, aswell as for screening expression libraries for the presence of anucleotide sequence encoding one or more of the proteins of theinvention or an antigenically related polypeptide. Antibodies furtherfind use as affinity ligands for purifying or isolating any one or moreof the proteins of the invention or an antigenically relatedpolypeptide. In embodiments, the antibody does not recognize (i.e.,specifically bind to) native Axmi205 and/or the parental Axmi205 toxinthat does not contain the modifications of the invention, and can beused to distinguish and/or separate an insecticidal protein of theinvention from native Axmi205.

Nucleic acids, Expression Cassettes, and Vectors.

As a further aspect, the invention provides nucleic acids encoding thepolypeptides of the invention, including modified polypeptides and toxinfragments as described herein.

According to some embodiments, the invention provides a nucleic acidmolecule comprising a nucleotide sequence that comprises, consistsessentially of, or consists of: (a) a nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQID NO: 43, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 69, SEQ ID NO: 73,SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ IDNO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105,SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ IDNO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123,SEQ ID NO: 125, SEQ ID NO: 127 or a toxin fragment thereof; (b) anucleotide sequence encoding an amino acid sequence that issubstantially identical to the amino acid sequence of (a); (c) anucleotide sequence that anneals under stringent hybridizationconditions to the nucleotide sequence of (a) or (b); or (d) a nucleotidesequence that differs from the nucleotide sequences of (a), (b) or (c)due to the degeneracy of the genetic code.

In embodiments, the nucleic acid molecule comprises a nucleotidesequence that comprises, consists essentially of, or consists of: (a) anucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQID NO: 44, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 70, SEQ ID NO: 74,SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 89, SEQ IDNO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO:108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, or SEQ IDNO: 126 or a toxin-encoding fragment thereof; (b) a nucleotide sequencethat is substantially identical to the nucleotide sequence of (a); (c) anucleotide sequence that anneals under stringent hybridizationconditions to the nucleotide sequence of (a) or (b); or (d) a nucleotidesequence that differs from the nucleotide sequences of (a), (b) or (c)due to the degeneracy of the genetic code. Optionally, the nucleotidesequence comprises, consists essentially of, or consists of thenucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQID NO: 44, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 70, SEQ ID NO: 74,SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 89, SEQ IDNO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO:108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, or SEQ IDNO: 126.

In embodiments, the nucleotide sequence is a partially or completelysynthetic sequence, e.g., that has codons optimized for expression in ahost organism, e.g., in a bacterium host or a plant host (for example, atransgenic monocot plant host or a transgenic dicot plant host).Non-limiting examples nucleotide sequences that are codon-optimized forexpression in a maize plant include SEQ ID NO: 75, SEQ ID NO: 76, andSEQ ID NO: 77.

In representative embodiments, for expression in transgenic plants, thenucleotide sequences of the invention are modified and/or optimized. Forexample, although in many cases genes from microbial organisms can beexpressed in plants at high levels without modification, low expressionin transgenic plants may result from microbial nucleotide sequenceshaving codons that are not preferred in plants. It is known in the artthat living organisms have specific preferences for codon usage, and thecodons of the nucleotide sequences described in this invention can bechanged to conform with plant preferences, while maintaining the aminoacids encoded thereby. Furthermore, it is known in the art that highexpression in plants, for example corn plants, can be achieved fromcoding sequences that have at least about 35% GC content, or at leastabout 45%, or at least about 50%, or at least about 60%. Microbialnucleotide sequences that have low GC contents may express poorly inplants. Although certain nucleotide sequences can be adequatelyexpressed in both monocotyledonous and dicotyledonous plant species,sequences can be modified to account for the specific codon preferencesand GC content preferences of monocotyledons or dicotyledons as thesepreferences have been shown to differ (Murray et al. Nucl. Acids Res.17:477-498 (1989)). In addition, in embodiments, the nucleotide sequenceis modified to remove illegitimate splice sites that may cause messagetruncation. Such modifications to the nucleotide sequences can be madeusing well known techniques of site directed mutagenesis, PCR, andsynthetic gene construction using the methods described, for example, inU.S. Pat. Nos. 5,625,136; 5,500,365 and 6,013,523.

In some embodiments, the invention provides synthetic coding sequencesor polynucleotide made according to the procedure disclosed in U.S. Pat.No. 5,625,136. In this procedure, maize preferred codons, i.e., thesingle codon that most frequently encodes that amino acid in maize, areused. The maize preferred codon for a particular amino acid can bederived, for example, from known gene sequences from maize. For example,maize codon usage for 28 genes from maize plants is found in Murray etal., Nucleic Acids Research 17:477-498 (1989). It is recognized thatcodons optimized for expression in one plant species will also functionin other plant species but possibly not at the same level as the plantspecies for which the codons were optimized. In this manner, thenucleotide sequences can be optimized for expression in any plant. It isrecognized that all or any part of a nucleotide sequence may beoptimized or synthetic. That is, a polynucleotide may comprise anucleotide sequence that is part native sequence and part codonoptimized sequence.

In representative embodiments, a polynucleotide of the invention is anisolated polynucleotide. In embodiments, a polynucleotide of theinvention is a recombinant polynucleotide.

In embodiments, the invention further provides a nucleic acid moleculecomprising a polynucleotide of the operably associated with a promoter(e.g., a heterologous promoter). Promoters can include, for example,constitutive, inducible, temporally regulated, developmentallyregulated, chemically regulated, tissue-preferred and/or tissue-specificpromoters. In particular aspects, a promoter useful with the inventionis a promoter capable of initiating transcription of a nucleotidesequence in a plant cell, e.g., in a cell of a monocot (e.g., maize orrice) or dicot (e.g., soybean, cotton) plant.

In embodiments, a heterologous promoter is a plant-expressible promoter(e.g., monocot expressible or dicot expressible). For example, withoutlimitation, the plant-expressible promoter can be selected from thefollowing promoters: ubiquitin, cestrum yellow virus, corn TrpA, OsMADS6, maize H3 histone, bacteriophage T3 gene 9 5′ UTR, corn sucrosesynthetase 1, corn alcohol dehydrogenase 1, corn light harvestingcomplex, corn heat shock protein, maize mtl, pea small subunit RuBPcarboxylase, rice actin, rice cyclophilin, Ti plasmid mannopinesynthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, beanglycine rich protein 1, potato patatin, lectin, CaMV 35S and S-E9 smallsubunit RuBP carboxylase promoter.

Although many promoters from dicotyledons have been shown to beoperational in monocotyledons and vice versa, in embodiments,dicotyledonous promoters are selected for expression in dicotyledons,and monocotyledonous promoters for expression in monocotyledons.However, there is no restriction to the provenance of selectedpromoters; it is sufficient that they are operational in driving theexpression of the nucleotide sequences in the desired cell.

The choice of promoter can vary depending on the temporal and spatialrequirements for expression, and also depending on the host cell to betransformed. Thus, for example, expression of the nucleotide sequencesof the invention can be in any plant and/or plant part, (e.g., inleaves, in stalks or stems, in ears, in inflorescences (e.g., spikes,panicles, cobs, etc.), in roots, seeds and/or seedlings, and the like).For example, where expression in a specific tissue or organ is desired,a tissue-specific or tissue-preferred promoter can be used (e.g., a rootspecific/preferred promoter). In contrast, where expression in responseto a stimulus is desired a promoter inducible by stimuli or chemicalscan be used. Where continuous expression at a relatively constant levelis desired throughout the cells of a plant a constitutive promoter canbe chosen.

Promoters useful with the invention include, but are not limited to,those that drive expression of a nucleotide sequence constitutively,those that drive expression when induced, and those that driveexpression in a tissue- or developmentally-specific manner. Thesevarious types of promoters are known in the art.

Suitable constitutive promoters include, for example, CaMV 35S promoter(Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter(see PCT Publication No. W004081173A2); maize Ubi 1 (Christensen et al.,Plant Mol. Biol. 18:675-689, 1992); rice actin (McElroy et al., PlantCell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet.81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462,1997); GOS2 (de Pater et al., Plant J November; 2(6):837-44, 1992);ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675-689, 1992); Ricecyclophilin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); MaizeH3 histone (Lepetit et al., Mol. Gen. Genet. 231: 276-285, 1992); Actin2 (An et al., Plant J. 10(1); 107-121, 1996), constitutive root tip CT2promoter (SEQ ID NO:1535; see also PCT application No. IL/2005/000627)and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995).Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026,5,608,149; 5,608,144; 5,604,121; 5,569,597: 5,466,785; 5,399,680;5,268,463; and 5,608,142.

Tissue-specific or tissue-preferential promoters useful for theexpression of the polypeptides of the invention in plants, optionallymaize, include those that direct expression in root, pith, leaf orpollen. Suitable tissue-specific promoters include, but not limited to,leaf-specific promoters (such as described, for example, by Yamamoto etal., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67,1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor etal., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol.23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA90:9586-9590, 1993), seed-preferred promoters (e.g., from seed specificgenes; Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al.,J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol.14: 633, 1990), Brazil Nut albumin (Pearson et al., Plant Mol. Biol. 18:235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214,1988), Glutelin (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986;Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke et al.,Plant Mol Biol, 143).323-32 1990), napA (Stalberg, et al., Planta 199:515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171-184, 1997),sunflower oleosin (Cummins, et al., Plant Mol. Biol. 19: 873-876,1992)], endosperm specific promoters (e.g., wheat LMW and HMW,glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b andg gliadins (EMB03:1409-15, 1984), Barley Itrl promoter, barley B1, C, Dhordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; MolGen Genet 250:750-60, 1996), Barley DOF (Mena et al., The Plant Journal,116(1): 53-62, 1998), Biz2 (EP99106056.7), Synthetic promoter(Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolaminNRP33, rice-globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8)885-889, 1998), rice alpha-globulin REB/OHP-1 (Nakase et al. Plant Mol.Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68,1997), maize ESR gene family (Plant J 12:235-46, 1997), sorgumgamma-kafirin (Plant Mol. Biol 32:1029-35, 1996)], embryo specificpromoters (e.g., rice OSH1; Sato et al., Proc. Nati. Acad. Sci. USA, 93:8117-8122), KNOX (Postma-Haarsma of al, Plant Mol. Biol. 39:257-71,1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)]flower-specific promoters, for example, AtPRP4, chalene synthase (chsA)(Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twellet al., Mol. Gen Genet. 217:240-245; 1989), apetala-3, and promotersspecific for plant reproductive tissues (e.g., OsMADS promoters; U.S.Patent Publication 2007/0006344).

Examples of promoters suitable for preferential expression in greentissue include many that regulate genes involved in photosynthesis andmany of these have been cloned from both monocotyledons anddicotyledons. One such promoter is the maize PEPC promoter from thephosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol.12:579-589 (1989)). Another promoter for root specific expression isthat described by de Framond (FEBS 290:103-106 (1991) or U.S. Pat. No.5,466,785). Another promoter useful in the invention is the stemspecific promoter described in U.S. Pat. No. 5,625,136, which naturallydrives expression of a maize trpA gene.

In addition, promoters functional in plastids can be used. Non-limitingexamples of such promoters include the bacteriophage T3 gene 9 5′ UTRand other promoters disclosed in U.S. Pat. No. 7,579,516. Otherpromoters useful with the invention include but are not limited to theS-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsininhibitor gene promoter (Kti3).

In some embodiments of the invention, inducible promoters can be used.Thus, for example, chemical-regulated promoters can be used to modulatethe expression of a gene in a plant through the application of anexogenous chemical regulator. Regulation of the expression of nucleotidesequences of the invention via promoters that are chemically regulatedenables the polypeptides of the invention to be synthesized only whenthe crop plants are treated with the inducing chemicals. Depending uponthe objective, the promoter may be a chemical-inducible promoter, whereapplication of a chemical induces gene expression, or achemical-repressible promoter, where application of the chemicalrepresses gene expression. Examples of such technology for chemicalinduction of gene expression is detailed in published application EP 0332 104 and U.S. Pat. No. 5,614,395.

Chemical inducible promoters are known in the art and include, but arenot limited to, the maize In2-2 promoter, which is activated bybenzenesulfonamide herbicide safeners, the maize GST promoter, which isactivated by hydrophobic electrophilic compounds that are used aspre-emergent herbicides, and the tobacco PR-1 a promoter, which isactivated by salicylic acid (e.g., the PR1a system), steroidsteroid-responsive promoters (see, e.g., the glucocorticoid-induciblepromoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88,10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see,e.g., Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, Lac repressor system promoters,copper-inducible system promoters, salicylate-inducible system promoters(e.g., the PR1a system), glucocorticoid-inducible promoters (Aoyama etal. (1997) Plant J. 11:605-612), and ecdysone-inducible systempromoters.

Other non-limiting examples of inducible promoters include ABA- andturgor-inducible promoters, the auxin-binding protein gene promoter(Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoidglycosyl-transferase promoter (Ralston et al. (1988) Genetics119:185-197), the MPI proteinase inhibitor promoter (Cordero et al.(1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphatedehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol.29:1293-1298; Martinez et al. (1989) J. Mol. Biol. 208:551-565; andQuigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are thebenzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) andalcohol-inducible (Intl Patent Application Publication Nos. WO 97/06269and WO 97/06268) systems and glutathione S-transferase promoters.Likewise, one can use any of the inducible promoters described in Gatz(1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev.Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically induciblepromoters useful for directing the expression of the nucleotidesequences of this invention in plants are disclosed in U.S. Pat. No.5,614,395. Chemical induction of gene expression is also detailed in EP0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395.

Another category of promoters useful in the invention are woundinducible promoters. Numerous promoters have been described that areexpressed at wound sites and also at the sites of phytopathogeninfection. Ideally, such a promoter should only be active locally at thesites of insect invasion, and in this way the insecticidal proteins onlyaccumulate in cells that need to synthesize the insecticidal proteins tokill the invading insect pest. Examples of promoters of this kindinclude those described by Stanford et al. Mol. Gen. Genet. 215:200-208(1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al.Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol.22:783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993),and Warner et al. Plant J. 3:191-201 (1993).

In embodiments a nucleic acid of the invention can comprise, consistessentially of, or consist of an expression cassette, or can becomprised within an expression cassette.

An expression cassette comprising a polynucleotide of interest may bechimeric, meaning that at least one of its components is heterologouswith respect to at least one other of its other components. Anexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.Typically, the expression cassette is heterologous with respect to thehost, i.e., the particular nucleic acid sequence of the expressioncassette does not occur naturally in the host cell and must have beenintroduced into the host cell or an ancestor of the host cell by atransformation event.

In addition to the promoters operatively associated with the nucleotidesequences of the invention, an expression cassette of this invention canalso include other regulatory elements. Regulatory elements include, butare not limited to, enhancers, introns, translation leader sequences,termination signals, and polyadenylation signal sequences. Examples ofsuitable transcription terminator signals are available and known in theart (e.g., tml from CaMV, E9 from rbcS). Any available terminator knownto function in plants can be used in the context of this invention.

Numerous other sequences can be incorporated into expression cassettesdescribed in this invention. These include sequences that have beenshown to enhance expression such as intron sequences (e.g., from Adhland bronzel) and viral leader sequences (e.g., from TMV, MCMV and AMV).

For more efficient initiation of translation, sequences adjacent to theinitiating methionine may be modified. For example, they can be modifiedby the inclusion of sequences known to be effective in plants. Joshi hassuggested an appropriate consensus for plants (NAR 15:6643-6653 (1987))and Clonetech suggests a further consensus translation initiator(1993/1994 catalog, page 210). These consensuses are suitable for usewith the nucleotide sequences of this invention. The sequences areincorporated into constructions comprising the nucleotide sequences, upto and including the ATG (while leaving the second amino acidunmodified), or alternatively up to and including the GTC subsequent tothe ATG (with the possibility of modifying the second amino acid of thetransgene).

In embodiments, it may be desired to target expression of thepolypeptides of the present invention to a specific cellular location inthe plant cell. In some cases, localization in the cytosol may bedesirable, whereas in other cases, localization in some subcellularorganelle may be preferred. Any mechanism for targeting gene products,e.g., in plants, can be used to practice this invention, and suchmechanisms are known to exist in plants and the sequences controllingthe functioning of these mechanisms have been characterized in somedetail. Sequences have been characterized which cause the targeting ofgene products to other cell compartments. For example, amino terminalsequences can be responsible for targeting a protein of interest to acell compartment, such as, a vacuole, mitochondrion, peroxisome, proteinbodies, endoplasmic reticulum, chloroplast, starch granule, amyloplast,apoplast or cell wall of a plant cell (e.g. Unger et. al. Plant Molec.Biol. 13: 411-418 (1989); Rogers et. al. (1985) Proc. Natl. Acad. Sci.USA 82: 6512-651; U.S. Pat. No. 7,102,057; WO 2005/096704. Optionally,the signal sequence may be an N-terminal signal sequence from waxy, anN-terminal signal sequence from gamma-zein, a starch binding domain, aC-terminal starch binding domain, a chloroplast targeting sequence,which imports the mature protein to the chloroplast (Comai et. al.(1988) J. Biol. Chem. 263: 15104-15109; van den Broeck, et. al. (1985)Nature 313: 358-363; U.S. Pat. No. 5,639,949) or a secretion signalsequence from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783(1990)). Additionally, amino terminal sequences in conjunction withcarboxyl terminal sequences are responsible for vacuolar targeting ofgene products and can be used with the present invention (Shinshi et.al. (1990) Plant Molec. Biol. 14: 357-368). In one embodiment, thesignal sequence selected includes the known cleavage site, and thefusion constructed takes into account any amino acids after the cleavagesite(s), which are required for cleavage. In some cases this requirementmay be fulfilled by the addition of a small number of amino acidsbetween the cleavage site and the transgene ATG or, alternatively,replacement of some amino acids within the transgene sequence. Theseconstruction techniques are well known in the art and are equallyapplicable to any cellular compartment.

It will be recognized that the above-described mechanisms for cellulartargeting can be utilized not only in conjunction with their cognatepromoters, but also in conjunction with heterologous promoters so as toeffect a specific cell-targeting goal under the transcriptionalregulation of a promoter that has an expression pattern different tothat of the promoter from which the targeting signal derives.

An expression cassette of the invention also can include a nucleotidesequence for a selectable marker, which can be used to select atransformed plant, plant part and/or plant cell. Many examples ofsuitable selectable markers are known in the art and can be used in theexpression cassettes described herein.

Examples of selectable markers include, but are not limited to, anucleotide sequence encoding neo or nptll, which confers resistance tokanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet.199:183-188); a nucleotide sequence encoding bar, which confersresistance to phosphinothricin; a nucleotide sequence encoding analtered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, whichconfers resistance to glyphosate (Hinchee et al. (1988) Biotech.6:915-922); a nucleotide sequence encoding a nitrilase such as bxn fromKlebsiella ozaenae that confers resistance to bromoxynil (Stalker et al.(1988) Science 242:419-423); a nucleotide sequence encoding an alteredacetolactate synthase (ALS) that confers resistance to imidazolinone,sulfonylurea or other ALS-inhibiting chemicals (EP Patent ApplicationNo. 154204); a nucleotide sequence encoding a methotrexate-resistantdihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem.263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenasethat confers resistance to dalapon; a nucleotide sequence encoding amannose-6-phosphate isomerase (also referred to as phosphomannoseisomerase (PMI)) that confers an ability to metabolize mannose (U.S.Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding analtered anthranilate synthase that confers resistance to 5-methyltryptophan; or a nucleotide sequence encoding hph that confersresistance to hygromycin. One of skill in the art is capable of choosinga suitable selectable marker for use in an expression cassette of thisinvention.

Additional selectable markers include, but are not limited to, anucleotide sequence encoding β-glucuronidase or uidA (GUS) that encodesan enzyme for which various chromogenic substrates are known; an R-locusnucleotide sequence that encodes a product that regulates the productionof anthocyanin pigments (red color) in plant tissues (Dellaporta et al.,“Molecular cloning of the maize R-nj allele by transposon-tagging withAc” 263-282 In: Chromosome Structure and Function: Impact of NewConcepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds.,Plenum Press 1988)); a nucleotide sequence encoding β-lactamase, anenzyme for which various chromogenic substrates are known (e.g., PADAC,a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci.USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes acatechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone, which in turncondenses to form melanin (Katz et al. (1983) J. Gen. Microbiol.129:2703-2714); a nucleotide sequence encoding β-galactosidase, anenzyme for which there are chromogenic substrates; a nucleotide sequenceencoding luciferase (lux) that allows for bioluminescence detection (Owet al. (1986) Science 234:856-859); a nucleotide sequence encodingaequorin which may be employed in calcium-sensitive bioluminescencedetection (Prasher et al. (1985) Biochem. Biophys. Res. Comm.126:1259-1268); or a nucleotide sequence encoding green fluorescentprotein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One ofskill in the art can choose a suitable selectable marker for use in anexpression cassette of this invention.

In some embodiments, an expression cassette of the invention also caninclude polynucleotides that encode other desired traits in addition tothe insecticidal proteins of the invention. Examples of such otherpolynucleotides include that those encode a polypeptide or a dsRNA forthe other desired trait(s) of interest. Such expression cassettescomprising the “stacked” traits may be used, e.g., to create plants,plant parts or plant cells having a desired phenotype with the stackedtraits (i.e., molecular stacking). Such stacked combinations in plantscan also be created by other methods including, but not limited to,cross breeding plants by any conventional methodology (i.e., a breedingstack). If stacked by genetically transforming the plants, thenucleotide sequences of interest can be combined at any time and in anyorder. For example, a transgenic plant comprising one or more desiredtraits can be used as the target to introduce further traits bysubsequent transformation. The additional nucleotide sequences can beintroduced simultaneously in a co-transformation protocol with anucleotide sequence, nucleic acid molecule, nucleic acid construct, orcomposition of this invention, provided by any combination of expressioncassettes. For example, if two nucleotide sequences will be introduced,they can be incorporated in separate cassettes (trans) or can beincorporated on the same cassette (cis). Expression of polynucleotidescan be driven by the same promoter or by different promoters. It isfurther recognized that polynucleotides can be stacked at a desiredgenomic location using a site-specific recombination system. See, e.g.,Intl Patent Application Publication Nos. WO 99/25821; WO 99/25854; WO99/25840; WO 99/25855 and WO 99/25853.

In representative embodiments, the expression cassette can also includean additional coding sequence for one or more polypeptides or doublestranded RNA molecules (dsRNA) of interest for an agronomic trait (e.g.,an agronomic trait that is primarily of benefit to a seed company,grower or grain processor). A polypeptide of interest can be anypolypeptide encoded by a nucleotide sequence of interest. Non-limitingexamples of polypeptides of interest that are suitable for production inplants include those resulting in agronomically important traits such asherbicide resistance (also sometimes referred to as “herbicidetolerance”), virus resistance, bacterial pathogen resistance, insectresistance, nematode resistance, or fungal resistance. See, e.g., U.S.Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. Inembodiments, the polypeptide of interest can be one that increases plantvigor or yield (including traits that allow a plant to grow at differenttemperatures, soil conditions and levels of sunlight and precipitation),or one that allows identification of a plant exhibiting a trait ofinterest (e.g., a selectable marker, seed coat color, etc.). Variouspolypeptides of interest, as well as methods for introducing thesepolypeptides into a plant, are described, for example, in U.S. Pat. Nos.4,761,373; 4,769,061; 4,810,648; 4,940,835; 4,975,374; 5,013,659;5,162,602; 5,276,268; 5,304,730; 5,495,071; 5,554,798; 5,561,236;5,569,823; 5,767,366; 5,879,903, 5,928,937; 6,084,155; 6,329,504 and6,337,431; as well as US Patent Publication No. 2001/0016956. See also,on the World Wide Web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.

Polynucleotides conferring resistance/tolerance to an herbicide thatinhibits the growing point or meristem, such as an imidazalinone or asulfonylurea can also be suitable in some embodiments of the invention.Exemplary polynucleotides in this category code for mutant ALS and AHASenzymes as described, e.g., in U.S. Pat. Nos. 5,767,366 and 5,928,937.U.S. Pat. Nos. 4,761,373 and 5,013,659 are directed to plants resistantto various imidazalinone or sulfonamide herbicides. U.S. Pat. No.4,975,374 relates to plant cells and plants containing a nucleic acidencoding a mutant glutamine synthetase (GS) resistant to inhibition byherbicides that are known to inhibit GS, e.g., phosphinothricin andmethionine sulfoximine. U.S. Pat. No. 5,162,602 discloses plantsresistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoicacid herbicides. The resistance is conferred by an altered acetylcoenzyme A carboxylase (ACCase).

Polypeptides encoded by nucleotides sequences conferring resistance toglyphosate are also suitable for the invention. See, e.g., U.S. Pat.Nos. 4,940,835 and 4,769,061. U.S. Pat. No. 5,554,798 disclosestransgenic glyphosate resistant maize plants, which resistance isconferred by an altered 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthasegene.

Polynucleotides coding for resistance to phosphono compounds such asglufosinate ammonium or phosphinothricin, and pyridinoxy or phenoxypropionic acids and cyclohexones are also suitable. See, European PatentApplication No. 0 242 246. See also, U.S. Pat. Nos. 5,879,903, 5,276,268and 5,561,236.

Other suitable polynucleotides include those coding for resistance toherbicides that inhibit photosynthesis, such as a triazine and abenzonitrile (nitrilase). See, U.S. Pat. No. 4,810,648. Additionalsuitable polynucleotides coding for herbicide resistance include thosecoding for resistance to 2,2-dichloropropionic acid, sethoxydim,haloxyfop, imidazolinone herbicides, sulfonylurea herbicides,triazolopyrimidine herbicides, s-triazine herbicides and bromoxynil.Also suitable are polynucleotides conferring resistance to a protoxenzyme, or that provide enhanced resistance to plant diseases; enhancedtolerance of adverse environmental conditions (abiotic stresses)including but not limited to drought, excessive cold, excessive heat, orexcessive soil salinity or extreme acidity or alkalinity; andalterations in plant architecture or development, including changes indevelopmental timing. See, e.g., U.S. Patent Publication No.2001/0016956 and U.S. Pat. No. 6,084,155.

Additional suitable polynucleotides include those coding for pesticidal(e.g., insecticidal) polypeptides. These polypeptides may be produced inamounts sufficient to control, for example, insect pests (i.e., insectcontrolling amounts). In embodiments, the polypeptide is alepidopteran-active, coleopteran-active, hem ipteran-active and/ordipteran-active polypeptide, or any combination thereof. It isrecognized that the amount of production of a pesticidal polypeptide ina plant to control insects or other pests may vary depending upon thecultivar, type of pest, environmental factors and the like.Polynucleotides useful for additional insect or other pest resistanceinclude, for example, those that encode toxins identified in Bacillusorganisms. Polynucleotides comprising nucleotide sequences encodingBacillus thuringiensis (Bt) Cry proteins from several subspecies havebeen cloned and recombinant clones have been found to be toxic tolepidopteran, dipteran and coleopteran insect larvae. Examples of suchBt insecticidal proteins include the Cry proteins such as Cry1Aa,Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A,Cry9B, Cry9C, the binary toxin Cry34/35, and modified Cry proteinsincluding Cry3Bb1, mCry3A, eCry3.1Ab., and the like, as well asvegetative insecticidal proteins such as Vip1, Vip2, Vip3, and the like,and any combination of the foregoing Bt insecticidal proteins. A fulllist of Bt-derived proteins can be found on the worldwide web at theBacillus thuringiensis Toxin Nomenclature Database maintained by theUniversity of Sussex (see also, Crickmore et al. (1998) Microbiol. Mol.Biol. Rev. 62:807-813).

In embodiments, an additional polypeptide is an insecticidal polypeptidederived from a non-Bt source, including without limitation, analpha-amylase, a peroxidase, a cholesterol oxidase, a patatin, aprotease, a protease inhibitor, a urease, an alpha-amylase inhibitor, apore-forming protein, a chitinase, a lectin, an engineered antibody orantibody fragment, a Bacillus cereus insecticidal protein, a Xenorhabdusspp. (such as X. nematophila or X. bovienii) insecticidal protein, aPhotorhabdus spp. (such as P. luminescens or P. asymobiotica)insecticidal protein, a Brevibacillus spp. (such as B. laterosporous)insecticidal protein, a Lysinibacillus spp. (such as L. sphearicus)insecticidal protein, a Chromobacterium spp. (such as C. subtsugae or C.piscinae) insecticidal protein, a Yersinia spp. (such as Y. entomophaga)insecticidal protein, a Paenibacillus spp. (such as P. propylaea)insecticidal protein, a Clostridium spp. (such as C. bifermentans)insecticidal protein, a Pseudomonas spp. (such as P. fluorescens) and alignin.

Also included are insecticidal traits based on RNA interference (RNAi).Double stranded RNA (dsRNA) molecules useful with the invention include,but are not limited to those that suppress target pest (e.g., insect)genes. In embodiments, the dsRNA targets a gene in a lepidopteran,coleopteran, hemipteran or dipteran insect pest, or any combination ofthe foregoing. As used herein the words “gene suppression”, when takentogether, are intended to refer to any of the well-known methods forreducing the levels of protein produced as a result of genetranscription to mRNA and subsequent translation of the mRNA. Genesuppression is also intended to mean the reduction of protein expressionfrom a gene or a coding sequence including posttranscriptional genesuppression and transcriptional suppression.

Posttranscriptional gene suppression is mediated by the homology betweenall or a part of a mRNA transcribed from a gene or coding sequencetargeted for suppression and the corresponding double stranded RNA usedfor suppression, and refers to the substantial and measurable reductionof the amount of available mRNA available in the cell for binding byribosomes. The transcribed RNA can be in the sense orientation to effectwhat is called co-suppression, in the anti-sense orientation to effectwhat is called anti-sense suppression, or in both orientations producinga dsRNA to effect what is called RNA interference (RNAi).Transcriptional suppression is mediated by the presence in the cell of adsRNA, a gene suppression agent, exhibiting substantial sequenceidentity to a promoter DNA sequence or the complement thereof to effectwhat is referred to as promoter trans suppression. Gene suppression maybe effective against a native plant gene associated with a trait, e.g.,to provide plants with reduced levels of a protein encoded by the nativegene or with enhanced or reduced levels of an affected metabolite. Genesuppression can also be effective against target genes in plant peststhat may ingest or contact plant material containing gene suppressionagents, specifically designed to inhibit or suppress the expression ofone or more homologous or complementary sequences in the cells of thepest. Such genes targeted for suppression can encode an essentialprotein, the predicted function of which is selected from the groupconsisting of muscle formation, juvenile hormone formation, juvenilehormone regulation, ion regulation and transport, digestive enzymesynthesis, maintenance of cell membrane potential, amino acidbiosynthesis, amino acid degradation, sperm formation, pheromonesynthesis, pheromone sensing, antennae formation, wing formation, legformation, development and differentiation, egg formation, larvalmaturation, digestive enzyme formation, haemolymph synthesis, haemolymphmaintenance, neurotransmission, cell division, energy metabolism,respiration, and apoptosis.

In embodiments, the dsRNA includes, without limitation, a dsRNAtargeting a vacuolar ATP synthase, a beta-tubulin, a 26S proteosomesubunit p28 protein, a EF1α 48D, a troponin I, a tetraspanin, agamma-coatomer, a beta-coatomer, and/or a juvenile hormone epoxidehydrolase. In embodiments, the polynucleotide encodes a DvSnf7 dsRNA(e.g., in corn event MON87411).

In some embodiments, the insecticidal protein of the invention and theadditional insect control agent are directed against the same targetinsect.

Polypeptides that are suitable for production in plants further includethose that improve or otherwise facilitate the conversion of harvestedplants or plant parts into a commercially useful product, including, forexample, increased or altered carbohydrate content or distribution,improved fermentation properties, increased oil content, increasedprotein content, improved digestibility, and increased nutraceuticalcontent, e.g., increased phytosterol content, increased tocopherolcontent, increased stanol content or increased vitamin content.Polypeptides of interest also include, for example, those resulting inor contributing to a reduced content of an unwanted component in aharvested crop, e.g., phytic acid, or sugar degrading enzymes. By“resulting in” or “contributing to” is intended that the polypeptide ofinterest can directly or indirectly contribute to the existence of atrait of interest (e.g., increasing cellulose degradation by the use ofa heterologous cellulase enzyme).

In some embodiments, the polypeptide contributes to improveddigestibility for food or feed. Xylanases are hem icellulolytic enzymesthat improve the breakdown of plant cell walls, which leads to betterutilization of the plant nutrients by an animal. This leads to improvedgrowth rate and feed conversion. Also, the viscosity of the feedscontaining xylan can be reduced. Heterologous production of xylanases inplant cells also can facilitate lignocellulosic conversion tofermentable sugars in industrial processing.

Numerous xylanases from fungal and bacterial microorganisms have beenidentified and characterized (see, e.g., U.S. Pat. No. 5,437,992;Coughlin et al. (1993) “Proceedings of the Second TRICEL Symposium onTrichoderma reesei Cellulases and Other Hydrolases” Espoo; Souminen andReinikainen, eds. (1993) Foundation for Biotechnical and IndustrialFermentation Research 8:125-135; U.S. Patent Publication No.2005/0208178; and PCT Publication No. WO 03/16654). In particular, threespecific xylanases (XYL-I, XYL-II, and XYL-III) have been identified inT. reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14:566;Torronen et al. (1992) Bio/Technology 10:1461; and Xu et al. (1998)Appl. Microbiol. Biotechnol. 49:718). In other embodiments, apolypeptide useful for the invention can be a polysaccharide degradingenzyme. Plants of this invention producing such an enzyme may be usefulfor generating, for example, fermentation feedstocks for bioprocessing.In some embodiments, enzymes useful for a fermentation process includealpha amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycotransferases, lipases,phytases, laccases, oxidases, esterases, cutinases, granular starchhydrolyzing enzyme and other glucoamylases.

Polysaccharide-degrading enzymes include: starch degrading enzymes suchas alpha-amylases (EC 3.2.1.1), glucuronidases (E.C. 3.2.1.131);exo-1,4-alpha-D glucanases such as amyloglucosidases and glucoamylase(EC 3.2.1.3), beta-amylases (EC 3.2.1.2), alpha-glucosidases (EC3.2.1.20), and other exo-amylases; starch debranching enzymes, such asa) isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41), and the like; b)cellulases such as exo-1,4-3-cellobiohydrolase (EC 3.2.1.91),exo-1,3-beta-D-glucanase (EC 3.2.1.39), beta-glucosidase (EC 3.2.1.21);c) L-arabinases, such as endo-1,5-alpha-L-arabinase (EC 3.2.1.99),alpha-arabinosidases (EC 3.2.1.55) and the like; d) galactanases such asendo-1,4-beta-D-galactanase (EC 3.2.1.89), endo-1,3-beta-D-galactanase(EC 3.2.1.90), alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC3.2.1.23) and the like; e) mannanases, such as endo-1,4-beta-D-mannanase(EC 3.2.1.78), beta-mannosidase (EC 3.2.1.25), alpha-mannosidase (EC3.2.1.24) and the like; f) xylanases, such as endo-1,4-beta-xylanase (EC3.2.1.8), beta-D-xylosidase (EC 3.2.1.37), 1,3-beta-D-xylanase, and thelike; and g) other enzymes such as alpha-L-fucosidase (EC 3.2.1.51),alpha-L-rhamnosidase (EC 3.2.1.40), levanase (EC 3.2.1.65), inulanase(EC 3.2.1.7), and the like. In one embodiment, the alpha-amylase is thesynthetic alpha-amylase, Amy797E, described is U.S. Pat. No. 8,093,453.

Further enzymes which may be used with the invention include proteases,such as fungal and bacterial proteases. Fungal proteases include, butare not limited to, those obtained from Aspergillus, Trichoderma, Mucorand Rhizopus, such as A. niger, A. awamori, A. oryzae and M. miehei. Insome embodiments, the polypeptides of this invention can becellobiohydrolase (CBH) enzymes (EC 3.2.1.91). In one embodiment, thecellobiohydrolase enzyme can be CBH1 or CBH2.

Other enzymes useful with the invention include, but are not limited to,hem icellulases, such as mannases and arabinofuranosidases (EC3.2.1.55); ligninases; lipases (e.g., E.C. 3.1.1.3), glucose oxidases,pectinases, xylanases, transglucosidases, alpha 1,6 glucosidases (e.g.,E.C. 3.2.1.20); esterases such as ferulic acid esterase (EC 3.1.1.73)and acetyl xylan esterases (EC 3.1.1.72); and cutinases (e.g., E.C.3.1.1.74).

In embodiments, the nucleic acids of the invention can further comprise,consist essentially of, or consist of a vector.

In embodiments, the polynucleotides and expression cassettes of theinvention are comprised within a vector. Vectors for use intransformation of plants and other organisms are well known in the art.Non-limiting examples of general classes of vectors include a plasmid,phage vector, phagemid vector, cosmid vector, fosmid, bacteriophage,artificial chromosome, or a viral vector. In embodiments, the vector isplant vector, e.g., for use in transformation of plants. In embodiments,the vector is a bacterial vector, e.g., for use in transformation ofbacteria. Suitable vectors for plants, bacteria and other organisms areknown in the art.

Transgenic Plants, Plant Parts, Plant Cells, Seed.

The invention also encompasses a transgenic non-human host cellcomprising a polynucleotide, a nucleic acid molecule, an expressioncassette, a vector, or a polypeptide of the invention. The transgenicnon-human host cell can include, but is not limited to, a plant cell(including a monocot cell and/or a dicot cell), a yeast cell, abacterial cell or an insect cell. Accordingly, in some embodiments, theinvention provides a bacterial cell selected from the genera Bacillus,Brevibacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteuria,Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella,Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,Azotobacter, Leuconostoc, or Alcaligenes. Thus, for example, asbiological insect control agents, the insecticidal proteins of theinvention can be produced by expression of a polynucleotide encoding thesame in a bacterial cell. For example, in some embodiments, a Bacillusthuringiensis cell comprising a polynucleotide encoding an insecticidalprotein of the invention is provided.

In embodiments, the transgenic plant cell is a dicot plant cell or amonocot plant cell. In additional embodiments, the dicot plant cell is asoybean cell, sunflower cell, tomato cell, cole crop cell, cotton cell,sugar beet cell or a tobacco cell. In further embodiments, the monocotcell is a barley cell, maize cell, oat cell, rice cell, sorghum cell,sugar cane cell or wheat cell. In embodiments, the invention provides aplurality of dicot cells or monocot cells comprising a polynucleotideexpressing an insecticidal protein of the invention. In embodiments, theplurality of cells are juxtaposed to form an apoplast and are grown innatural sunlight. In embodiments, the transgenic plant cell isnon-propagating and/or cannot regenerate a whole plant.

In embodiments of the invention, an insecticidal protein of theinvention is expressed in a higher organism, for example, a plant. Inthis case, transgenic plants expressing effective amounts of theinsecticidal protein protect themselves from plant pests such as insectpests. When an insect starts feeding on such a transgenic plant, itingests the expressed insecticidal protein. This can deter the insectfrom further biting into the plant tissue or may even harm or kill theinsect. In embodiments, a polynucleotide of the invention is insertedinto an expression cassette, which is then stably integrated in thegenome of the plant. In other embodiments, the polynucleotide isincluded in a non-pathogenic self-replicating virus.

In some embodiments of the invention, a transgenic plant cell comprisinga nucleic acid molecule or polypeptide of the invention is a cell of aplant part, a plant organ or a plant culture (each as described herein)including, but not limited to, a root, a leaf, a seed, a flower, afruit, a pollen cell, organ or plant culture, and the like, or a calluscell or culture.

A transgenic plant or plant cell in accordance with the invention may bea monocot or dicot plant or plant cell and includes, but is not limitedto, corn (maize), soybean, rice, wheat, barley, rye, oat, sorghum,millet, sunflower, safflower, sugar beet, cotton, sugarcane, oilseedrape, alfalfa, tobacco, peanut, vegetable (including, sweet potato,bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip,carrot, eggplant, cucumber, radish, spinach, potato, tomato, asparagus,onion, garlic, melon, pepper, celery, squash, pumpkin, zucchini, and thelike), fruit (including, apple, pear, quince, plum, cherry, peach,nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple,avocado, papaya, mango, banana, and the like), a specialty plant orplant cell (such as Arabidopsis), or a woody plant or plant cell (suchas coniferous and/or deciduous trees). In embodiments, a plant or plantcell of the of the invention is a crop plant or plant cell such asmaize, sorghum, wheat, sunflower, tomato, a crucifer, pepper, potato,cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, oilseedrape plant or plant cell, and the like.

The invention further provides a part of a transgenic plant of theinvention. Optionally, the plant part comprises an insecticidal proteinof the invention and/or a nucleic acid encoding the same.

The invention further provides a seed of a transgenic plant of theinvention or a seed that produces the transgenic plant of the invention.Optionally, the seed comprises an insecticidal protein of the inventionand/or a nucleic acid encoding the same.

Additional embodiments of the invention include harvested productsproduced from the transgenic plants, plant parts or seed of theinvention, as well as a processed product produced from a harvestedproduct. A harvested product can be a whole plant or any plant part, asdescribed herein. Thus, in some embodiments, non-limiting examples of aharvested product include a seed, a fruit, a flower or part thereof(e.g., an anther, a stigma, and the like), a leaf, a stem, and the like.In other embodiments, a processed product includes, but is not limitedto, a flour, meal, oil, starch, cereal, and the like produced from aharvested seed or other plant part of the invention. Optionally, theharvested product or the processed product comprises an insecticidalprotein of the invention and/or a nucleic acid encoding the same.

In other embodiments, the invention provides an extract from atransgenic plant, plant part or of the invention, optionally wherein theextract comprises an insecticidal protein of the invention and/or anucleic acid encoding the same. Extracts from plants or plant parts canbe made according to procedures well known in the art (See, de la Torreet al., Food, Agric. Environ. 2(1):84-89 (2004); Guidet, Nucleic AcidsRes. 22(9): 1772-1773 (1994); Lipton et al., Food Agric. Immun.12:153-164 (2000)).

The insecticidal protein of the invention can function in the plantpart, plant cell, plant organ, seed, harvested product, processedproduct or extract, and the like, as an insect control agent. In otherwords, the insecticidal protein can continue to perform the insecticidalfunction it had in the transgenic plant. The nucleic acid can functionto express the insecticidal protein. As an alternative to encoding theinsecticidal protein of the invention, the nucleic acid can function toidentify a transgenic plant part, plant cell, plant organ, seed,harvested product, processed product or extract of the invention.

In embodiments, a transgenic plant, plant part, plant cell, plant organ,or seed of the invention is hem izygous for a polynucleotide orexpression cassette of the invention. In embodiments, a transgenicplant, plant part, plant cell, plant organ, or seed of the invention ishomozygous for a polynucleotide or expression cassette of the invention.

In embodiments, a transgenic plant, plant part, plant cell, plant organ,seed, harvested product, processed product or extract has increasedresistance to one or more insect pests (e.g., a coleopteran pest, suchas a corn rootworm, for example, WCRW) as compared with a suitablecontrol that does not comprise a nucleic acid encoding an insecticidalprotein of the invention.

Plant Transformation.

Procedures for transforming plants are well known and routine in the artand are described throughout the literature. Non-limiting examples ofmethods for transformation of plants include transformation viabacterial-mediated nucleic acid delivery (e.g., via Agrobacterium),viral-mediated nucleic acid delivery, silicon carbide or nucleic acidwhisker-mediated nucleic acid delivery, liposome mediated nucleic aciddelivery, microinjection, microparticle bombardment,calcium-phosphate-mediated transformation, cyclodextrin-mediatedtransformation, electroporation, nanoparticle-mediated transformation,sonication, infiltration, PEG-mediated nucleic acid uptake, as well asany other electrical, chemical, physical (mechanical) or biologicalmechanism that results in the introduction of nucleic acid into theplant cell, including any combination thereof. General guides to variousplant transformation methods known in the art include Miki et al.(“Procedures for Introducing Foreign DNA into Plants” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) andRakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

For Agrobacterium-mediated transformation, binary vectors or vectorscarrying at least one T-DNA border sequence are generally suitable,whereas for direct gene transfer (e.g., particle bombardment and thelike) any vector is suitable and linear DNA containing only theconstruction of interest can be used. In the case of direct genetransfer, transformation with a single DNA species or co-transformationcan be used (Schocher et al., Biotechnology 4:1093-1096 (1986)). Forboth direct gene transfer and Agrobacterium-mediated transfer,transformation is usually (but not necessarily) undertaken with aselectable marker that may be a positive selection (e.g., PhosphomannoseIsomerase), provide resistance to an antibiotic (e.g., kanamycin,hygromycin or methotrexate) or a herbicide (e.g., glyphosate orglufosinate). However, the choice of selectable marker is not criticalto the invention.

Agrobacterium-mediated transformation is a commonly used method fortransforming plants because of its high efficiency of transformation andbecause of its broad utility with many different species.Agrobacterium-mediated transformation typically involves transfer of thebinary vector carrying the foreign DNA of interest to an appropriateAgrobacterium strain that may depend on the complement of vir genescarried by the host Agrobacterium strain either on a co-resident Tiplasmid or chromosomally (Uknes et al. (1993) Plant Cell 5:159-169). Thetransfer of the recombinant binary vector to Agrobacterium can beaccomplished by a triparental mating procedure using Escherichia colicarrying the recombinant binary vector, a helper E. coli strain thatcarries a plasmid that is able to mobilize the recombinant binary vectorto the target Agrobacterium strain. Alternatively, the recombinantbinary vector can be transferred to Agrobacterium by nucleic acidtransformation (Höfgen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

Dicots as well as monocots may be transformed using Agrobacterium.Methods for Agrobacterium-mediated transformation of rice include wellknown methods for rice transformation, such as those described in any ofthe following: European patent application EP 1198985 A1, Aldemita andHodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3):491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), whichdisclosures are incorporated by reference herein as if fully set forth.In the case of corn transformation, the preferred method is as describedin either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frameet al. (Plant Physiol 129(1): 13-22, 2002), which disclosures areincorporated by reference herein as if fully set forth. Said methods arefurther described by way of example in B. Jenes et al., Techniques forGene Transfer, in: Transgenic Plants, Vol. 1, Engineering andUtilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991)205-225). The nucleic acids or the construct to be expressed ispreferably cloned into a vector, which is suitable for transformingAgrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. AcidsRes. 12 (1984) 8711). Agrobacteria transformed by such a vector can thenbe used in known manner for the transformation of plants, such as plantsused as a model, like Arabidopsis or crop plants such as, by way ofexample, tobacco plants, for example by immersing bruised leaves orchopped leaves in an Agrobacterial solution and then culturing them insuitable media. The transformation of plants by means of Agrobacteriumtumefaciens is described, for example, by Hagen and Willmitzer in Nucl.Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White,Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol.1, Engineering and Utilization, eds. S. D. Kung and R. Wu, AcademicPress, 1993, pp. 15-38.

Transformation of a plant by recombinant Agrobacterium usually involvesco-cultivation of the Agrobacterium with explants from the plant andfollows methods well known in the art. Transformed tissue is regeneratedon selection medium carrying an antibiotic or herbicide resistancemarker between the binary plasmid T-DNA borders.

As discussed previously, another method for transforming plants, plantparts and plant cells involves propelling inert or biologically activeparticles at plant tissues and cells. See, e.g., U.S. Pat. Nos.4,945,050; 5,036,006 and 5,100,792. Generally, this method involvespropelling inert or biologically active particles at the plant cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the nucleic acid ofinterest. Alternatively, a cell or cells can be surrounded by the vectorso that the vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., a dried yeast cell, a driedbacterium or a bacteriophage, each containing one or more nucleic acidssought to be introduced) also can be propelled into plant tissue.

In other embodiments, a polynucleotide of the invention can be directlytransformed into the plastid genome. A major advantage of plastidtransformation is that plastids are generally capable of expressingbacterial genes without substantial modification, and plastids arecapable of expressing multiple open reading frames under control of asingle promoter. Plastid transformation technology is extensivelydescribed in U.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, in PCTapplication no. WO 95/16783, and in McBride et al. (1994) Proc. Nati.Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplasttransformation involves introducing regions of cloned plastid DNAflanking a selectable marker together with the gene of interest into asuitable target tissue, e.g., using biolistics or protoplasttransformation (e.g., calcium chloride or PEG mediated transformation).The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitatehomologous recombination with the plastid genome and thus allow thereplacement or modification of specific regions of the plastome.Initially, point mutations in the chloroplast 16S rRNA and rps12 genesconferring resistance to spectinomycin or streptomycin can be utilizedas selectable markers for transformation (Svab, Z., Hajdukiewicz, P.,and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub,J. M., and Maliga, P. (1992) Plant Cell 4, 39-45). The presence ofcloning sites between these markers allows creation of a plastidtargeting vector for introduction of foreign genes (Staub, J. M., andMaliga, P. (1993) EMBO J. 12, 601-606). Substantial increases intransformation frequency can be obtained by replacement of the recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial aadA gene encoding the spectinomycin-cletoxifyingenzyme aminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this markerhad been used successfully for high-frequency transformation of theplastid genome of the green alga Chlamydomonas reinhardtii(Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15-20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by homologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In one embodiment, apolynucleotide of the invention can be inserted into a plastid-targetingvector and transformed into the plastid genome of a desired plant host.Thus, plants homoplastic for plastid genomes containing a nucleotidesequence of the invention can be obtained, which are capable of highexpression of the polynucleotide.

Methods of selecting for transformed, transgenic plants, plant cells orplant tissue culture are routine in the art and can be employed in themethods of the invention provided herein. For example, a recombinantvector of the invention also can include an expression cassettecomprising a nucleotide sequence for a selectable marker, which can beused to select a transformed plant, plant part or plant cell.

Further, as is well known in the art, intact transgenic plants can beregenerated from transformed plant cells, plant tissue culture orcultured protoplasts using any of a variety of known techniques. Plantregeneration from plant cells, plant tissue culture or culturedprotoplasts is described, for example, in Evans et al. (Handbook ofPlant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983));and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants,Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)).

Additionally, the genetic properties engineered into the transgenicseeds and plants, plant parts, or plant cells of the invention describedabove can be passed on by sexual reproduction or vegetative growth andtherefore can be maintained and propagated in progeny plants. Generally,maintenance and propagation make use of known agricultural methodsdeveloped to fit specific purposes such as harvesting, sowing ortilling.

A polynucleotide therefore can be introduced into the plant, plant partor plant cell in any number of ways that are well known in the art, asdescribed above. Therefore, no particular method for introducing one ormore polynucleotides into a plant is relied upon, rather any method thatallows the one or more polynucleotides to be stably integrated into thegenome of the plant can be used. Where more than one polynucleotides isto be introduced, the respective polynucleotides can be assembled aspart of a single nucleic acid molecule, or as separate nucleic acidmolecules, and can be located on the same or different nucleic acidmolecules. Accordingly, the polynucleotides can be introduced into thecell of interest in a single transformation event, in separatetransformation events, or, for example, in plants, as part of a breedingprotocol.

Once a desired polynucleotide has been transformed into a particularplant species, it may be propagated in that species or moved into othervarieties of the same species, particularly including commercialvarieties, using traditional breeding techniques. Furthermore, anAxmi205 transgene can be modified in situ to incorporate themodifications of the present invention using genome editing techniques.

Pesticidal Compositions.

In embodiments, the invention provides an insecticidal compositioncomprising an insecticidal protein of the invention in an agriculturallyacceptable carrier. As used herein an “agriculturally-acceptablecarrier” can include natural or synthetic, organic or inorganic materialwhich is combined with the active protein to facilitate its applicationto or in the plant, or part thereof. Examples of agriculturallyacceptable carriers include, without limitation, powders, dusts,pellets, granules, sprays, emulsions, colloids, and solutions.Agriculturally-acceptable carriers further include, but are not limitedto, inert components, dispersants, surfactants, adjuvants, tackifiers,stickers, binders, or combinations thereof, that can be used inagricultural formulations. Such compositions can be applied in anymanner that brings the pesticidal proteins or other pest control agentsin contact with the pests. Accordingly, the compositions can be appliedto the surfaces of plants or plant parts, including seeds, leaves,flowers, stems, tubers, roots, and the like. In other embodiments, aplant producing an insecticidal protein of the invention in planta is anagriculturally-acceptable carrier of the expressed insecticidal protein.In embodiments, the compositions and agriculturally-acceptable carriersof the invention exclude transgenic plants.

In further embodiments, the insecticidal composition comprises abacterial cell or a transgenic bacterial cell of the invention, whereinthe bacterial cell or transgenic bacterial cell produces an insecticidalprotein of the invention. Such an insecticidal composition can beprepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof Bacillus thuringiensis (Bt), including a transgenic Bt culture. Inembodiments, a composition of the invention may comprise at least about1%, at least about 5%, at least about 10%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,or at least 99% by weight a polypeptide of the invention. In additionalembodiments, the composition comprises from about 1% to about 99% byweight of the insecticidal protein of the invention.

The insecticidal proteins of the invention can be used in combinationwith other pest control agents (e.g., insect control agents) to increasethe target pest (e.g., insect) spectrum and/or for the prevention ormanagement of insect resistance. Furthermore, the use of theinsecticidal proteins of the invention in combination with aninsecticidal agent which has a different mode of action and/or targets adifferent receptor in the insect gut has particular utility for theprevention and/or management of insect resistance. In embodiments, theinsecticidal protein of the invention is used in combination withanother insect control agent that targets the same insect pest.

Therefore, in some embodiments, the invention provides a compositionthat controls one or more plant pests (e.g., an insect pest such as alepidopteran insect pest, a coleopteran insect pest, a hem ipteraninsect pest and/or a dipteran insect pest), wherein the compositioncomprises a first pest control agent, which is an insecticidal proteinof the invention and at least a second pest control agent (e.g., aninsect control agent) that is different from the first pest controlagent. In other embodiments, the composition is a formulation fortopical application to a plant. In still other embodiments, thecomposition is a transgenic plant. In further embodiments, thecomposition is a combination of a formulation topically applied to atransgenic plant. In some embodiments, the formulation comprises thefirst pest control agent, which is an insecticidal protein of theinvention when the transgenic plant comprises the second pest controlagent. In other embodiments, the formulation comprises the second pestcontrol agent when the transgenic plant comprises the first pest controlagent, which is an insecticidal protein of the invention.

In some embodiments, the second pest control agent can be one or more ofa chemical pesticide, such as an insecticide, a Bt insecticidal protein,and/or a non-Bt pesticidal agent including without limitation aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, aBrevibacillus laterosporus insecticidal protein, a Bacillus sphaericusinsecticidal protein, a protease inhibitor (both serine and cysteinetypes), a lectin, an alpha-amylase, a peroxidase, a cholesterol oxidase,or a double stranded RNA (dsRNA) molecule.

In other embodiments, the second pest control agent is one or morechemical pesticides, which is optionally a seed coating. Non-limitingexamples of chemical pesticides include pyrethroids, carbamates,neonicotinoids, neuronal sodium channel blockers, insecticidalmacrocyclic lactones, gamma-aminobutyric acid (GABA) antagonists,insecticidal ureas and juvenile hormone mimics. In other embodiments,the chemical pesticide is one or more of abamectin, acephate,acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin,azinphos-methyl, bifenthrin, binfenazate, buprofezin, carbofuran,chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl,chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin,lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin,diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan,emamectin, endosulfan, esfenvalerate, ethiprole, fenothicarb,fenoxycarb, fenpropathrin, fenproximate, fenvalerate, fipronil, flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701), flufenoxuron,fonophos, halofenozide, hexaflumuron, imidacloprid, indoxacarb,isofenphos, lufenuron, malathion, metaldehyde, methamidophos,methidathion, methomyl, methoprene, methoxychlor, monocrotophos,methoxyfenozide, nithiazin, novaluron, noviflumuron (XDE-007), oxamyl,parathion, parathion-methyl, permethrin, phorate, phosalone, phosmet,phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl,pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060), sulprofos,tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos,thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin,trichlorfon and triflumuron, aldicarb, oxamyl, fenamiphos, amitraz,chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor,etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate,hexythiazox, propargite, pyridaben and tebufenpyrad. In still otherembodiments, the chemical pesticide is selected from one or more ofcypermethrin, cyhalothrin, cyfluthrin and beta-cyfluthrin,esfenvalerate, fenvalerate, tralomethrin, fenothicarb, methomyl, oxamyl,thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb,spinosad, abamectin, avermectin, emamectin, endosulfan, ethiprole,fipronil, flufenoxuron, triflumuron, diofenolan, pyriproxyfen,pymetrozine and amitraz.

In additional embodiments, the second pest control agent can be one ormore of any number of Bt insecticidal proteins including but not limitedto a Cry protein, a vegetative insecticidal protein (VIP) andinsecticidal chimeras of any of the preceding insecticidal proteins. Inother embodiments, the second pest control agent is a Cry proteinselected from: Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Af, Cry1Ag,Cry1Ah, Cry1Ai, Cry1Aj, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Bd, Cry1Be, Cry1Bf,Cry1Bg, Cry1Bh, Cry1Bi, Cry1Ca, Cry1Cb, Cry1Da, Cry1db, Cry1Dc, Cry1Dd,Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Gb, Cry1Gc, Cry1Ha, Cry1Hb,Cry1Hc, Cry1Ia, Cry1Ib, Cry1Ic, Cry1Id, Cry1Ie, Cry1If, Cry1Ig, Cry1Ja,Cry1Jb, Cry1Jc, Cry1Jd, Cry1Ka, Cry1La, Cry1Ma, Cry1Na, Cry1Nb, Cry2Aa,Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Aj,Cry2Ak, Cry2Al, Cry2Ba, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry4Aa, Cry4Ba,Cry4Ca, Cry4Cb, Cry4Cc, Cry5Aa, Cry5Ab, Cry5Ac, Cry5Ad, Cry5Ba, Cry5Ca,Cry5Da, Cry5Ea, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry7Ac, Cry7Ba, Cry7Bb,Cry7Ca, Cry7Cb, Cry7Da, Cry7Ea, Cry7Fa, Cry7Fb, Cry7Ga, Cry7Gb, Cry7Gc,Cry7Gd, Cry7Ha, Cry7Ia, Cry7Ja, Cry7Ka, Cry7Kb, Cry7La, Cry8Aa, Cry8Ab,Cry8Ac, Cry8Ad, Cry8Ba, Cry8Bb, Cry8Bc, Cry8Ca, Cry8Da, Cry8db, Cry8Ea,Cry8Fa, Cry8Ga, Cry8Ha, Cry8Ia, Cry8Ib, Cry8Ja, Cry8Ka, Cry8Kb, Cry8La,Cry8Ma, Cry8Na, Cry8Pa, Cry8Qa, Cry8Ra, Cry8Sa, Cry8Ta, Cry9Aa, Cry9Ba,Cry9Bb, Cry9Ca, Cry9Da, Cry9db, Cry9Dc, Cry9Ea, Cry9Eb, Cry9Ec, Cry9Ed,Cry9Ee, Cry9Fa, Cry9Ga, Cry10Aa, Cry11Aa, Cry11Ba, Cry11Bb, Cry12Aa,Cry13Aa, Cry14Aa, Cry14Ab, Cry15Aa, Cry16Aa, Cry17Aa, Cry18Aa, Cry18Ba,Cry18Ca, Cry19Aa, Cry19Ba, Cry19Ca, Cry20Aa, Cry20Ba, Cry21Aa, Cry21Ba,Cry21Ca, Cry21Da, Cry21Ea, Cry21Fa, Cry21Ga, Cry21Ha, Cry22Aa, Cry22Ab,Cry22Ba, Cry22Bb, Cry23Aa, Cry24Aa, Cry24Ba, Cry24Ca, Cry25Aa, Cry26Aa,Cry27Aa, Cry28Aa, Cry29Aa, Cry29Ba, Cry30Aa, Cry30Ba, Cry30Ca, Cry30Da,Cry30db, Cry30Ea, Cry30Fa, Cry30Ga, Cry31Aa, Cry31Ab, Cry31Ac, Cry31Ad,Cry32Aa, Cry32Ab, Cry32Ba, Cry32Ca, Cry32Cb, Cry32Da, Cry32Ea, Cry32Eb,Cry32Fa, Cry32Ga, Cry32Ha, Cry32Hb, Cry32Ia, Cry32Ja, Cry32Ka, Cry32La,Cry32Ma, Cry32Mb, Cry32Na, Cry32Oa, Cry32Pa, Cry32Qa, Cry32Ra, Cry32Sa,Cry32Ta, Cry32Ua, Cry33Aa, Cry34Aa, Cry34Ab, Cry34Ac, Cry34Ba, Cry35Aa,Cry35Ab, Cry35Ac, Cry35Ba, Cry36Aa, Cry37Aa, Cry38Aa, Cry39Aa, Cry40Aa,Cry40Ba, Cry40Ca, Cry40Da, Cry41Aa, Cry41Ab, Cry41Ba, Cry42Aa, Cry43Aa,Cry43Ba, Cry43Ca, Cry43Cb, Cry43Cc, Cry44Aa, Cry45Aa, Cry46Aa Cry46Ab,Cry47Aa, Cry48Aa, Cry48Ab, Cry49Aa, Cry49Ab, Cry50Aa, Cry50Ba, Cry51Aa,Cry52Aa, Cry52Ba, Cry53Aa, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa,Cry56Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba, Cry60Aa, Cry60Ba,Cry61Aa, Cry62Aa, Cry63Aa, Cry64Aa, Cry65Aa, Cry66Aa, Cry67Aa, Cry68Aa,Cry69Aa, Cry69Ab, Cry70Aa, Cry70Ba, Cry70Bb, Cry71Aa, Cry72Aa, Cry73Aa,or any combination of the foregoing. In embodiments, the Cry protein isa mCry3A protein (e.g., in corn event MIR604), a eCry3.1Ab protein(e.g., in corn event 5307), a Cry3Bb1 protein (e.g., in corn eventMON88017) and/or a Cry34/35Ab1 binary protein (e.g., in corn eventDAS-59122).

In further embodiments, the second pest control agent is one or moreVip3 vegetative insecticidal proteins selected from Vip3Aa1, Vip3Aa2,Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vip3Aa8, Vip3Aa9, Vip3Aa10,Vip3Aa11, Vip3Aa12, Vip3Aa13, Vip3Aa14, Vip3Aa15, Vip3Aa16, Vip3Aa17,Vip3Aa18, Vip3Aa19, Vip3Aa20, Vip3Aa21, Vip3Aa22, Vip3Aa2, Vip3Aa24,Vip3Aa25, Vip3Aa26, Vip3Aa27, Vip3Aa28, Vip3Aa29, Vip3Aa30, Vip3Aa31,Vip3Aa32, Vip3Aa33, Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38,Vip3Aa39, Vip3Aa40, Vip3Aa41, Vip3Aa42, Vip3Aa43, Vip3Aa44, Vip3Ab1,Vip3Ab2, Vip3Ac1, Vip3Ad1, Vip3Ad2, Vip3Ae1, Vip3Af1, Vip3Af2, Vip3Af3,Vip3Ag1,Vip3Ag2,Vip3Ag3 HM117633, Vip3Ag4, Vip3Ag5, Vip3Ah1, Vip3Ba1,Vip3Ba2, Vip3Bb1, Vip3Bb2, Vip3Bb3, or any combination of the foregoing.In embodiments, the Vip3 protein is Vip3Aa (U.S. Pat. No. 6,137,033),for example, as represented by corn event MIR162 (U.S. Pat. Nos.8,232,456; 8,455,720; and 8,618,272).

In embodiments, the second pest control agent may be derived fromsources other than B. thuringiensis. For example, the second pestcontrol agent can be an alpha-amylase, a peroxidase, a cholesteroloxidase, a patatin, a protease, a protease inhibitor, a urease, analpha-amylase inhibitor, a pore-forming protein, a chitinase, a lectin,an engineered antibody or antibody fragment, a Bacillus cereusinsecticidal protein, a Xenorhabdus spp. (such as X. nematophila or X.bovienii) insecticidal protein, a Photorhabdus spp. (such as P.luminescens or P. asymobiotica) insecticidal protein, a Brevibacillusspp. (such as B. laterosporous) insecticidal protein, a Lysinibacillusspp. (such as L. sphearicus) insecticidal protein, a Chromobacteriumspp. (such as C. subtsugae or C. piscinae) insecticidal protein, aYersinia spp. (such as Y. entomophaga) insecticidal protein, aPaenibacillus spp. (such as P. propylaea) insecticidal protein, aClostridium spp. (such as C. bifermentans) insecticidal protein, aPseudomonas spp. (such as P. fluorescens) and a lignin. In otherembodiments, the second agent may be at least one insecticidal proteinderived from an insecticidal toxin complex (Tc) from Photorhabdus,Xenorhabus, Serratia, or Yersinia. In other embodiments. Theinsecticidal protein may be an ADP-ribosyltransferase derived from aninsecticidal bacteria, such as Photorhabdus ssp. In other embodiments,the insecticidal protein may be a non-Bt VIP protein, such as VIP1and/or VIP2 from B. cereus. In still other embodiments, the insecticidalprotein may be a binary toxin derived from an insecticidal bacteria,such as ISP1A and ISP2A from B. laterosporous or BinA and BinB from L.sphaericus. In still other embodiments, the insecticidal protein may beengineered or may be a hybrid or chimera of any of the precedinginsecticidal proteins.

In some embodiments, the second pesticidal agent can benon-proteinaceous, for example, an interfering RNA molecule such as adsRNA, which can be expressed transgenically or applied as part of acomposition (e.g., using topical methods). An interfering RNA typicallycomprises at least a RNA fragment against a target gene, a spacersequence, and a second RNA fragment which is complementary to the first,so that a double-stranded RNA structure can be formed. RNA interference(RNAi) occurs when an organism recognizes double-stranded RNA (dsRNA)molecules and hydrolyzes them. The resulting hydrolysis products aresmall RNA fragments of about 19-24 nucleotides in length, called smallinterfering RNAs (siRNAs). The siRNAs then diffuse or are carriedthroughout the organism, including across cellular membranes, where theyhybridize to mRNAs (or other RNAs) and cause hydrolysis of the RNA.Interfering RNAs are recognized by the RNA interference silencingcomplex (RISC) into which an effector strand (or “guide strand”) of theRNA is loaded. This guide strand acts as a template for the recognitionand destruction of the duplex sequences. This process is repeated eachtime the siRNA hybridizes to its complementary-RNA target, effectivelypreventing those mRNAs from being translated, and thus “silencing” theexpression of specific genes from which the mRNAs were transcribed.Interfering RNAs are known in the art to be useful for insect control(see, for example, publication WO2013/192256, incorporated by referenceherein). An interfering RNA designed for use in insect control producesa non-naturally occurring double-stranded RNA, which takes advantage ofthe native RNAi pathways in the insect to trigger down-regulation oftarget genes that may lead to the cessation of feeding and/or growth andmay result in the death of the insect pest. The interfering RNA moleculemay confer insect resistance against the same target pest as the proteinof the invention, or may target a different pest. The targeted insectplant pest may feed by chewing, sucking, or piercing. Interfering RNAsare known in the art to be useful for insect control. In embodiments,the dsRNA useful for insect control is described in U.S. ProvisionalApplication Nos. 62/371,259, 62/371,261, or 62/371,262, filed on Aug. 5,2016. In embodiments, the dsRNA useful for insect control is describedin U.S. Pat. Nos. 7,812,219; 9,238,822; 9,340,797; 8,946,510; or USpatent publication US2014/0275208. In embodiments, the dsRNA useful forinsect control is described in U.S. patent application Ser. Nos.12/868,994; 14/207,313; or 14/207,318. In embodiments, the dsRNA targetsa gene encoding a vacuolar ATP synthase, a beta-tubulin, a 26Sproteosome subunit p28 protein, a EF1α 48D, a troponin I, a tetraspanin,a gamma-coatomer, a beta-coatomer, and/or a juvenile hormone epoxidehydrolase. In embodiments, the dsRNA is a DvSnf7 dsRNA (e.g., in cornevent MON87411).

In embodiments, the interfering RNA confers resistance against anon-insect plant pest, such as a nematode pest or a virus pest.

In still further embodiments, the first insect control agent, which isan insecticidal protein of the invention and the second pest controlagent are co-expressed in a transgenic plant. This co-expression of morethan one pesticidal principle in the same transgenic plant can beachieved by genetically engineering a plant to contain and express thenucleic acid sequences encoding the insect control agents. For example,the co-expression of more than one pesticidal agent in the sametransgenic plant can be achieved by making a single recombinant vectorcomprising coding sequences of more than one pesticidal agent in a“molecular stack” and genetically engineering a plant to contain andexpress all the pesticidal agents in the transgenic plant. Suchmolecular stacks may be also be made by using mini-chromosomes asdescribed, for example in U.S. Pat. No. 7,235,716. Alternatively, aplant, Parent 1, can be genetically engineered for the expression of theinsecticidal protein of the invention. A second plant, Parent 2, can begenetically engineered for the expression of a second pest controlagent. By crossing Parent 1 with Parent 2, progeny plants are obtainedwhich express both insect control agents from Parents 1 and 2 (i.e., abreeding stack).

In other embodiments, the invention provides a stacked transgenic plantresistant to plant pest infestation comprising a nucleic acid (e.g.,DNA) sequence encoding a dsRNA for suppression of an essential gene in atarget pest and a nucleic acid e.g., (DNA) sequence encoding aninsecticidal protein of the invention exhibiting insecticidal activityagainst the target pest. It has been reported that dsRNAs areineffective against certain lepidopteran pests (Rajagopol et al. 2002.J. Biol. Chem. 277:468-494), likely due to the high pH of the midgutwhich destabilizes the dsRNA. Therefore, in some embodiments where thetarget pest is a lepidopteran pest, an insecticidal protein of theinvention may act to transiently reduce the midgut pH which serves tostabilize the co-ingested dsRNA rendering the dsRNA effective insilencing the target genes.

Transgenic plants or seed comprising and/or expressing an insecticidalprotein of the invention can also be treated with an insecticide orinsecticidal seed coating as described in U.S. Pat. Nos. 5,849,320 and5,876,739. In embodiments, where both the insecticide or insecticidalseed coating and the transgenic plant or seed of the invention areactive against the same target insect, for example a coleopteran pest(e.g., a corn rootworm, for example, WCRW), the combination is useful(i) in a method for further enhancing activity of the composition of theinvention against the target insect, and/or (ii) in a method forpreventing development of resistance to the composition of the inventionby providing yet another mechanism of action against the target insect.Thus, in embodiments, the invention provides a method of enhancingcontrol of a coleopteran insect population comprising providing atransgenic plant or seed of the invention and applying to the plant orthe seed an insecticide or insecticidal seed coating to a transgenicplant or seed of the invention.

Even where the insecticide or insecticidal seed coating is activeagainst a different insect, the insecticide or insecticidal seed coatingis useful to expand the range of insect control, for example by addingan insecticide or insecticidal seed coating that has activity againstlepidopteran insects to a transgenic seed of the invention, which, insome embodiments, has activity against coleopteran insects, the coatedtransgenic seed produced controls both lepidopteran and coleopteraninsect pests.

Methods of Making and Using the Insecticidal Proteins, Nucleic Acids,and Transgenic Plants.

The invention also encompasses methods of producing an insect-resistant(e.g., a coleopteran insect-resistant) transgenic plant. Inrepresentative embodiments, the method comprises: introducing into aplant a polynucleotide, expression cassette or vector of the inventioncomprising a nucleotide sequence that encodes an insecticidal protein ofthe invention (including toxin fragments and modified forms that aresubstantially identical to the polypeptides specifically disclosedherein), wherein the nucleotide sequence is expressed in the plant toproduce the insecticidal protein of the invention, thereby conferring tothe plant resistance to the insect pest, and producing aninsect-resistant transgenic plant (e.g., as compared with a suitablecontrol plant, such as a plant that does not comprise thepolynucleotide, expression cassette or vector of the invention and/ordoes not express a polypeptide of the invention).

In embodiments, the method of introducing the polynucleotide, expressioncassette or vector of the invention into the plant comprises firsttransforming a plant cell with the polynucleotide, expression cassetteor vector and regenerating a transgenic plant therefrom, where thetransgenic plant comprises the polynucleotide, expression cassette orvector and expresses the insecticidal protein of the invention.

Alternatively, or additionally, the introducing step can comprisecrossing a first plant comprising the polynucleotide, expressioncassette or vector with a second plant (e.g., a different plant from thefirst plant, for example, a plant that does not comprise thepolynucleotide, expression cassette or vector) and, optionally,producing a progeny plant that comprises the polynucleotide, expressioncassette or vector and expresses an insecticidal protein of theinvention, thereby resulting in increased resistance to at least oneinsect pest. Thus, a transgenic plant of the invention encompasses aplant that is the direct result of a transformation event and theprogeny thereof (of any generation) that comprise the polynucleotide,expression cassette or vector and optionally expresses the insecticidalprotein resulting in increased resistance to at least one insect pest.

As a further option, genome editing techniques can be used to modify insitu a transgene encoding a native Axmi205 protein (SEQ ID NO: 1) or avariant Axmi205 (for example, the Axmi205 variants described inWO2013/016617 to Athenix; and US 2014/0274885 A1 to Pioneer Hi-Bred) toincorporate the mutations of the invention. To illustrate, in a plantcomprising a heterologous polynucleotide sequence encoding a nativeAxmi205 protein or a variant thereof, the coding sequence of the nativeor variant Axmi205 can be further modified in situ by targeted DNAediting using various genome editing techniques such as zinc fingernucleases (ZNFs), transcription activator-like effector nucleases(TALENS), meganucleases and Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) (U.S. Pat. No. 8,697,359; Ran et al.). TheCRISPR system can be used to introduce specific nucleotide modificationsat the target sequence. Originally discovered in bacteria, where severaldifferent CRISPR cascades function as innate immune systems and naturaldefense mechanisms, the engineered CRISPR-Cas9 system can be programmedto target specific stretches of genetic code and to make cuts at preciselocations. Over the past few years, those capabilities have beenharnessed and used as genome editing tools, enabling researchers topermanently modify genes in plant cells.

Thus, the invention encompasses methods for generating a polynucleotideencoding a modified Axmi205 protein of the invention wherein said methodcomprises modifying a plant genome comprising a polynucleotide encodinga native Axmi205 protein (e.g., SEQ ID NO: 1) or an Axmi205 variantusing a gene editing technique such as CRISPR to incorporate one or moremutations according to the present invention. In embodiments, the methodinvolves targeting of Cas9 to the specific genomic locus, in this case anative Axmi205 protein-encoding polynucleotide or a polynucleotideencoding an Axmi205 variant, via a 20 nt guide sequence of thesingle-guide RNA. An online CRISPRO Design Tool can identify suitabletarget sites (on the world wide web at tools.genome-engineering.org; Ranet al., Genome engineering using the CRISPR-Cas9 system. NatureProtocols I; 2281-2308 (2013)). Target plants for the mutagenesis/genomeediting methods according to the invention are any monocot or dicotplant (each as described further herein) into which a polynucleotideencoding a native Axmi205 protein or Axmi205 mutant protein has beenintroduced.

Optionally, the methods of producing a transgenic plant with increasedresistance to an insect pest (e.g., a coleopteran insect pest, such asWCRW) further comprises obtaining a progeny plant for one or moregenerations, wherein the progeny plant comprises the polynucleotide, thenucleic acid molecule or the vector and has increased resistance to acoleopteran insect pest.

The invention further provides a method of identifying a transgenicplant of the invention, the method comprising detecting the presence ofa polynucleotide, expression cassette, vector or insecticidal protein ofthe invention in a plant (or a plant cell, plant part, and the likederived therefrom), and thereby identifying the plant as a transgenicplant of the invention based on the presence of the polynucleotide,expression cassette, vector or insecticidal protein of the invention.

The invention further provides a method of producing a transgenic plantwith increased resistance to at least one insect pest (e.g., a least onecoleopteran pest), the method comprising: planting a seed comprising apolynucleotide, expression cassette or vector of the invention, andgrowing a transgenic plant from the seed, where the transgenic plantcomprises the polynucleotide, expression cassette or vector and producesthe insecticidal protein.

In embodiments, transgenic plants produced by the methods of theinvention comprise a polynucleotide, expression cassette or vector ofthe invention. In embodiments, a transgenic plant produced by themethods of the invention comprise an insecticidal protein of theinvention and, optionally have increased resistance to at least oneinsect pest.

The methods of producing a transgenic plant described herein optionallycomprise a further step of harvesting a seed from the transgenic plant,where the seed comprises the polynucleotide, expression cassette orvector and produces the insecticidal protein. Optionally, the seedproduces a further transgenic plant that comprises the polynucleotide,expression cassette or vector and produces the insecticidal protein, andthereby has increased resistance to at least one insect pest.

The invention further provides plant parts, plant cells, plant organs,plant cultures, seed, plant extracts, harvested products and processedproducts of the transgenic plants produced by the methods of theinvention.

As a further aspect, the invention also provides a method of producingseed, the method comprising: providing a transgenic plant that comprisesa polynucleotide, expression cassette or vector of the invention, andharvesting a seed from the transgenic plant, wherein the seed comprisesthe polynucleotide, expression cassette, vector and produces theinsecticidal protein. Optionally, the seed produces a further transgenicplant that comprises the polynucleotide, expression cassette or vectorand produces the insecticidal protein, and thereby has increasedresistance to at least one insect pest. In representative embodiments,the step of providing the transgenic plant comprises planting a seedthat produces the transgenic plant.

The invention further provides a method of producing a hybrid plantseed, the method comprising: crossing a first inbred plant, which is atransgenic plant comprising a polynucleotide, expression cassette orvector of the invention, and optionally expressing an insecticidalprotein of the invention with a different inbred plant (e.g., an inbredplant that does not comprise a polynucleotide, expression cassette orvector of the invention) and allowing hybrid seed to form. Optionally,the method further comprises harvesting a hybrid seed. In embodiments,the hybrid seed comprises the polynucleotide, expression cassette orvector of the invention, and in embodiments may further comprise aninsecticidal protein of the invention and have increased resistance toan insect pest. In embodiments, the hybrid seed produces a transgenicplant that comprises the polynucleotide, expression cassette or vectorof the invention, expresses the insecticidal protein of the invention,and has increased resistance to at least one insect pest.

In some embodiments, a transgenic plant of the invention controls atleast one coleopteran insect pest (as described herein). In embodiments,the transgenic plant controls a corn rootworm insect pest or colony(e.g., a WCRW insect pest or colony) that is resistant to a mCry3Aprotein (e.g., in corn event MIR604), a eCry3.1Ab protein (e.g., in cornevent 5307), a Cry3Bb1 protein (e.g., in corn event MON88017), aCry34/35Ab1 binary protein (e.g., in corn event DAS-59122) and/or a RNAitrait, such as DvSnf7 dsRNA (e.g., in corn event MON87411).

In further embodiments, a method of controlling at least one insect pest(e.g., at least one coleopteran insect pest, such as a corn rootworm,for example, WCRW) comprises providing an insecticidal protein of theinvention. In embodiments, the method comprises delivering (e.g., orallydelivering) to the insect pest or an environment thereof an effectiveamount of an insecticidal protein of the invention. Generally, to beeffective, the polypeptide is orally ingested by the insect. However,the insecticidal protein can be delivered to the insect in manyrecognized ways. The ways to deliver a protein orally to an insectinclude, but are not limited to, providing the protein (1) in atransgenic plant, wherein the insect eats (ingests) one or more parts ofthe transgenic plant, thereby ingesting the polypeptide that isexpressed in the transgenic plant; (2) in a formulated proteincomposition(s) that can be applied to or incorporated into, for example,insect growth media; (3) in a protein composition(s) that can be appliedto the surface, for example, sprayed, onto the surface of a plant part,which is then ingested by the insect as the insect eats one or more ofthe sprayed plant parts; (4) a bait matrix; or (5) any otherart-recognized protein delivery system. Thus, any method of oraldelivery to an insect can be used to deliver the toxic proteins of theinvention. In some particular embodiments, the insecticidal protein ofthe invention is delivered orally to an insect, for example, where theinsect ingests one or more parts of a transgenic plant of the invention.

In other embodiments, the insecticidal protein of the invention isdelivered orally to an insect, wherein the insect ingests one or moreparts of a plant sprayed with a composition comprising the insecticidalprotein of the invention. Delivering the composition of the invention toa plant surface can be done using any method known to those of skill inthe art for applying compounds, compositions, formulations and the liketo plant surfaces. Some non-limiting examples of delivering to orcontacting a plant or part thereof include spraying, dusting,sprinkling, scattering, misting, atomizing, broadcasting, soaking, soilinjection, soil incorporation, drenching (e.g., root, soil treatment),dipping, pouring, coating, leaf or stem infiltration, side dressing orseed treatment, and the like, and combinations thereof. These and otherprocedures for contacting a plant or part thereof with a compound(s),composition(s) or formulation(s) are well-known to those of skill in theart.

In further embodiments, the invention provides a method of controlling acoleopteran insect pest that is resistant to a mCry3A protein (e.g., incorn event MIR604), a eCry3.1Ab protein (e.g., in corn event 5307), aCry3Bb1 protein (e.g., in corn event MON88017), a Cry34/35Ab1 binaryprotein (e.g., in corn event DAS-59122) and/or a RNAi trait, such asDvSnf7 dsRNA (e.g., in corn event MON87411), the method comprisingdelivering to the coleopteran insect or an environment thereof aneffective amount of an insecticidal protein or composition of theinvention. In representative embodiments, the resistant insect pest is aresistant corn rootworm (e.g., WCRW) insect pest or colony.

In other embodiments, the invention provides a method of reducing orpreventing the development of resistance in a population of a targetcoleopteran insect pest to a mCry3A protein (e.g., in corn eventMIR604), a eCry3.1Ab protein (e.g., in corn event 5307), a Cry3Bb1protein (e.g., in corn event MON88017), a Cry34/35Ab1 binary protein(e.g., in corn event DAS-59122) and/or a RNAi trait, such as DvSnf7dsRNA (e.g., in corn event MON87411) expressed in a transgenic plant,the method comprising delivering to the target coleopteran insectpopulation a transgenic plant comprising a polynucleotide comprising anucleotide sequence encoding a mCry3A protein, a nucleotide sequenceencoding a eCry3.1Ab protein, a nucleotide sequence encoding a Cry3Bb1protein, a nucleotide sequence(s) encoding a Cry34/35Ab1 binary proteinand/or a nucleotide sequence encoding a RNAi trait; and apolynucleotide, expression cassette or vector of the inventionexpressing an insecticidal protein of the invention. In someembodiments, the target coleopteran insect pest is a corn rootworm(e.g., WCRW). According to foregoing embodiments, the transgenic plantcan comprise a breeding stack of two or more of the insecticidal traits,a molecular stack of two or more of the insecticidal traits, or acombination of both.

In some embodiments, the invention encompasses a method of providing afarmer with a means of controlling an insect pest (e.g., a coleopteranpest, such as a corn rootworm, for example, WCRW), the method comprisingsupplying or selling to the farmer plant material such as a seed, theplant material comprising a polynucleotide, expression cassette, vectorcapable of expressing an insecticidal protein of the invention. Inembodiments, the plant material comprises the insecticidal protein ofthe invention and, optionally, has increased resistance to at least oneinsect pest. In embodiments, the plant material is a seed, and a plantgrown from the seed comprises the polynucleotide, expression cassette orvector of the invention, expresses an insecticidal protein of theinvention, and has increased resistance to the at least one insect pest.

In addition to providing compositions, the invention provides methods ofproducing an insecticidal protein toxic to a coleopteran pest. Such amethod comprises, culturing a transgenic non-human host cell thatcomprises a polynucleotide, expression cassette or vector of theinvention that expresses an insecticidal protein of the invention underconditions in which the host cell produces the insecticidal protein thatis toxic to the coleopteran pest. In some embodiments, the transgenicnon-human host cell is a plant cell. In some other embodiments, theplant cell is a maize cell. In other embodiments, the conditions underwhich the plant cell or maize cell are grown include natural sunlight.In other embodiments, the transgenic non-human host cell is a bacterialcell. In still other embodiments, the transgenic non-human host cell isa yeast cell.

In embodiments, the methods of the invention provide control of at leastone coleopteran, lepidopteran, dipteran, hemipteran, orthopteran and/orthysanopteran insect pest (each as described in more detail herein). Inembodiments, the insecticidal protein is active against a coleopteranpest, including without limitation: Diabrotica spp. (for example,Diabrotica barberi (northern corn rootworm); D. virgifera virgifera(western corn rootworm); D. undecimpunctata howardii (southern cornrootworm); D. balteata (banded cucumber beetle); D. undecimpunctataundecimpunctata (western spotted cucumber beetle); D. significata(3-spotted leaf beetle); D. speciosa (chrysanthemum beetle); D.virgifera zeae (Mexican corn rootworm); D. beniensis; D. cristata, D.curviplustalata; D. dissimilis; D. elegantula; D. emorsitans; D.graminea; D. hispanloe; D. lemniscata; D. linsleyi; D. milleri; D.nummularis; D. occlusal; D. porrecea; D. scutellata; D. tibialis; D.trifasciata and D. viridula; and any combination thereof), Leptinotarsaspp. (for example, L. decemlineata; Colorado potato beetle); Chrysomelaspp. (for example, C. scripta; cottonwood leaf beetle); Hypothenemusspp. (for example, H. hampei; coffee berry borer); Sitophilus spp. (forexample, S. zeamais; maize weevil); Epitrix spp. (for example, E.hirtipennis [tobacco flea beetle] and E. cucumeris [potato fleabeetle]); Phyllotreta spp. (for example, P. cruciferae [crucifer fleabeetle] and P. pusilla [western black flea beetle]; Anthonomus spp. (forexample, A. grandis [boll weevil]) and A. eugenii [pepper weevil]);Hemicrepidus spp. (for example, H. memnonius; wireworms); Melanotus spp.(for example, M. communis; wireworm); Ceutorhychus spp. (for example, C.assimilis; cabbage seedpod weevil); Phyllotreta spp. (for example, P.cruciferae; crucifer flea beetle); Aeolus spp. (for example, A.mellillus; wireworm); Aeolus spp. (for example, A. mancus; wheatwireworm); Horistonotus spp. (for example, H. uhlerii; sand wireworm);Sphenophorus spp. (for example, S. maidis [maize billbug], S. zeae[timothy billbug], S. parvulus [bluegrass billbug], and S. callosus[southern corn billbug]); Phyllophaga spp. (for example, white grubs);Chaetocnema spp. (for example, C. pulicaria; corn flea beetle); Popilliaspp. (for example, P. japonica; Japanese beetle); Epilachna spp. (forexample, E. varivestis; Mexican bean beetle); Cerotoma spp. (forexample, C. trifurcate; Bean leaf beetle); Epicauta spp. (for example,E. pestifera and E. lemniscata; Blister beetles); and any combination ofthe foregoing.

In embodiments, the methods of the invention provide control of a cornrootworm (e.g., WCRW) insect pest or colony that is resistant to amCry3A protein (e.g., in corn event MIR604), a eCry3.1Ab protein (e.g.,in corn event 5307), a Cry3Bb1 protein (e.g., in corn event MON88017), aCry34/35Ab1 binary protein (e.g., in corn event DAS-59122) and/or a RNAitrait, such as DvSnf7 dsRNA (e.g., maize event MON87411).

The invention also provides for uses of the insecticidal proteins,nucleic acids, transgenic plants, plant parts, seed and insecticidalcompositions of the invention, for example, to control an insect pest,such as a coleopteran pest (as described herein).

In embodiments, the invention provides a method of using apolynucleotide, expression cassette, vector or host cell of theinvention to produce an insecticidal composition for controlling aninsect pest (e.g., a coleopteran insect pest).

In embodiments, the invention provides a method of using apolynucleotide, expression cassette or vector of the invention toproduce a transgenic seed, where the transgenic seed grows a transgenicplant with increased resistance to an insect pest.

As another aspect, the invention also contemplates the use of atransgenic plant of the invention to produce a transgenic seed, which isoptionally a hybrid seed.

In embodiments, the invention provides a method of using an insecticidalprotein, polynucleotide, expression cassette, vector, transgenic plantor insecticidal composition of the invention to prevent the developmentof resistance in a population of a target coleopteran insect pest to amCry3A protein (e.g., in corn event MIR604), a eCry3.1Ab protein (e.g.,in corn event 5307), a Cry3Bb1 protein (e.g., in corn event MON88017), aCry34/35Ab1 binary protein (e.g., in corn event DAS-59122) and/or a RNAitrait such as DvSnf7 dsRNA (e.g., maize event MON87411).

The invention will now be described with reference to the followingexamples. It will be appreciated by those skilled in the art that theseexamples do not limit the scope of the claims to the invention, but arerather intended to be exemplary of certain embodiments. Otherembodiments of the invention may be practiced without departing from thespirit and the scope of the invention, the scope of which is defined bythe disclosure and the appended claims.

EXAMPLE 1 Mutant Selection

Axmi205 is an insecticidal protein, isolated from Chromobacteriumpiscinae and previously described in U.S. Pat. No. 8,575,425 B2 andSampson et al. (Discovery of a novel insecticidal protein fromChromobacterium piscinae, with activity against Western Corn Rootworm,Diabrotica virgifera virgifera, J. Invertebrate Pathology 142: 34-43(2016)). See also GenBank Accession No. AML23188.1. The amino acid andcDNA sequences of native Axmi205 are provided as SEQ ID NO: 1 and SEQ IDNO: 2, respectively.

Previous experiments had determined that the native amino acid sequenceof Axmi205 (SEQ ID NO: 1) showed delayed digestion in a standardSimulated Gastric Fluid (SGF) assay, with an undigested 23 kDa bandstill present at the 30 minutes time period, rendering this toxin lessdesirable from a regulatory standpoint. The SGF assay is used toapproximate the digestion of the protein in the mammalian gut, and is astandard component of the evaluation of any new insecticidal protein forregulatory approval.

Mass spectrometry analysis identified the 23 kDa band as consisting ofthe entire second domain of the protein, corresponding approximately toresidues 303-526 of the native Axmi205 protein (SEQ ID NO: 1). Sites oflikely pepsin cleavage in the native Axmi205 protein were predictedusing published guidelines for pepsin cleavage at pH <1.3 (Keil, B.Specificity of proteolysis. Springer-Verlag Berlin-Heidelberg-New York,pp. 335. (1992)). Stretches of the native Axmi205 protein withoutpredicted pepsin cleavage sites were identified, and residues withinthese regions were selected for mutational analysis to increaseproteolytic cleavage of the protein (e.g., by pepsin) while maintaininginsecticidal activity. For example, in some mutants, one or morehydrophobic amino acids were substituted or inserted into the nativeAxmi205 protein sequence. In other cases, cysteine residues werereplaced to reduce the possibility of disulfide bond formation, possiblyopening up the tertiary protein structure to facilitate enzyme access.See FIG. 1 .

EXAMPLE 2 Production of Native Axmi205 and Engineered Axmi205 Variants

Expression vector pEBDuet28A containing the native Axmi205 cDNA sequence(SEQ ID NO: 2) with a Tobacco Etch Virus (TEV) protease removableN-terminal 10× His tag was used as the starting molecule for allengineered Axmi205 (eAxmi205) mutant production. A DNA fragmentcontaining the Axmi205 mutations was synthesized and subsequently clonedinto the EcoRI-SalI sites of the Axmi205 source vector.

Vectors expressing the eAxmi205 variants were transformed into E. coliBL21*(DE3) (INVITROGEN™) for protein production. Briefly, 100 mLcultures of Terrific Broth media were grown at 37° C. until mid-logphase (OD₆₀₀=0.6-1.0), upon which protein expression was induced using0.1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) and subsequentlytransferred to 18° C. overnight. Cells were harvested by centrifugationand lysed using mechanical lysis. Bacterial lysates were clarified andpassed over His GRAVITRAP™ (GE Healthcare) nickel columns to isolate theprotein of interest. Unbound proteins were removed via washing, and theeAxmi205 variants were then eluted using imidazole. Protein-containingfractions were pooled and dialyzed against 1×PBS overnight at 4° C. Theprotein concentration was determined, and the protein was aliquoted andsnap frozen in liquid nitrogen for long term storage at −80° C.

EXAMPLE 3 Simulated Gastric Fluid T10 Protocol

As a preliminary screen, purified eAxmi205 mutants were tested fordigestability after 10 minutes (T10) of exposure to simulated gastricfluid (SGF). Briefly, test proteins were normalized to a commonconcentration (typically ˜1 mg/m L) to allow for a single stock of SGFto be produced at the proper ratof pepsin to test protein (approximately1111 U pepsin/mL, in G-Con solution {2 mg/mL NaCl, pH 1.2}). Thedigestion reaction was initiated by adding 30 μL of test protein to 270μL SGF at 37° C. At 10 minutes (T10), 100 μL of the test protein-SGFreaction mixture was removed and the reaction terminated by adding thetest mixture aliquot to 100 μL of preheated (95° C.) Stop Solutioncomprised of 65% Tricine Loading Buffer (Bio-Rad 2× Tricine Load Bufferw/10% β-mercaptoethanol) and 35% 500 mM NaHCO₃ pH 11.0. A time zero (T0)data point was produced by adding 10 μL of test protein to 100 μL ofpreheated (95° C.) Stop Solution and 95 uL of SGF. All samples wereheated at 95° C. for 5 minutes, and then stored on ice until SDS-PAGEanalysis.

For SDS-PAGE, 30 μL of each reaction was loaded on a 10-20% Tris-tricinepeptide gel (Bio-Rad) prior to standard protein gel electrophoresis.After electrophoresis for 20 minutes, the Tris-tricine gel wasimmediately fixed with a 40% methanol:10% acetic acid mixture. The gelwas then stained with GELCODE™ Blue protein stain (ThermoFisherScientific) for 1 hour at room temperature. After 1 hour, the gel wasde-stained with distilled water overnight.

On SDS-PAGE, the native Axmi205 retains a band at approximately 23 kDaat T10. Mutants were identified in which no undigested fragments wereobserved that were larger than approximately 3-4 kDa at T10, indicatingthat these eAxmi205 mutants are likely to exhibit more completedigestion by pepsin as compared with the native Axmi205 protein, andwere selected for further evaluation of insecticidal bioactivity and amore complete SGF digestion profile.

EXAMPLE 4 eAxmi205 Bioactivity Against Western Corn Rootworm

eAxmi205 mutants that showed loss of the ˜23 kDa band at 10 minutes inthe preliminary SGF testing (Example 2) were then evaluated forbioactivity against Western Corn Rootworm (WCRW; Diabrotica virgiferavirgifera) larvae in an artificial diet bioassay.

Samples of purified protein for each mutant were normalized to 0.4 mg/mL(200 μg/mL final) and combined with an equal volume of heated artificialinsect diet (Bioserv, Inc., Frenchtown N.J.) in 1.5 mL centrifuge tubesand then applied to small petri-dishes. After the diet-sample mixturecooled and solidified, 12 WCRW newly hatched larvae were added to eachdish, and the dish was subsequently sealed. Petri dishes were kept atambient conditions with regard to temperature, lighting and relativehumidity. Protein storage buffer (i.e., PBS) was used as a negativecontrol, and native Axmi205 used as the positive control. Mortality wasassessed on day 3 or 4 and again on day 6.

eAxmi205 mutants showing bioactivity to WCRW were subjected to a morecomplete evaluation for digestion in SGF.

EXAMPLE 5 SGF Time Course Assay

A full time course of SGF digestion was conducted on mutants that bothpassed the initial SGF T10 test (Example 2) and retained bioactivity toWCRW (Example 3).

In summary, solid pepsin (˜3 mg, ˜3640 U/mg solid) was dissolved in 1 mLG-Con solution (2 mg/mL NaCl, pH 1.2) to create a concentrated stock ofpepsin. The pepsin stock was diluted to the appropriate concentration inG-Con solution to make 10 U pepsin/μg protein (determined by the initialtest protein stock concentration), to produce the final SGF solution foruse in the assay. The digestion reaction was initiated (T0) by adding 70μL of the test protein stock to 630 μL of the SGF solution at 37° C. Thereaction was stopped by removing 100 μL of the reaction mixture at 1, 2,5,10, 30 and 60 minutes (i.e., T1, T2, T5, T10, T30 and T60) and mixingwith 100 μL Stop Solution (65% Tricine Loading buffer with 5%β-mercaptoethanol, 35% 500 mM NaHCO₃ pH 11.0) preheated to 95° C.Controls include a T0 control (10 μL test protein+100 μL StopSolution+90 μL SGF), a SGF control (90 μL SGF+10 μL buffer+100 μL StopSolution at T0 and T60), and a test protein control (90 μL G-Consolution+10 μL test protein+100 μL Stop Solution at T0 and T60). Afteradding the reaction mixture aliquot to the Stop Solution, all sampleswere heated at 95° C. for 5 minutes, and then stored on ice untilSDS-PAGE analysis.

For SDS-PAGE, 30 μL of each reaction was loaded on a 10-20% Tris-tricinepeptide gel (Bio-Rad) prior to standard protein gel electrophoresis.After 20 minutes of electrophoresis, the Tris-tricine gel wasimmediately fixed with a 40% methanol:10% acetic acid mixture. The gelwas then stained with GELCODE™ Blue protein stain (ThermoFisherScientific) for 1 hour at room temperature. After 1 hour, the gel wasde-stained with distilled water overnight.

Standard Western analysis was also done with a polyclonal rabbitanti-Axmi205 antibody to identify immunoreactive bands.

EXAMPLE 6 Synthesis, Expression and Purification of eAxmi205 Mutants

Thirty-six eAxmi205 mutants that were synthesized and purified arelisted in Table 1. Mutants numbered 1 to 30 are substitution mutants,and mutants numbered 31 to 36 are insertion mutants. The amino acidpositions of the eAxmi205 mutations indicated in the following tablesand discussion are all with reference to the native Axmi205 proteinsequence of SEQ ID NO: 1.

TABLE 1 eAxmi205 Mutations SEQ ID SEQ ID # eAxmi205 Mutant Amino AcidCoding Sequence 1 K328Y 3 4 2 K328L 5 6 3 K328F 7 8 4 Y404F 9 10 5 K402F11 12 6 K402N 13 14 7 K402L 15 16 8 Y404F + K402L 17 18 9 R416L 19 20 10P386L 21 22 11 R391L 23 24 12 R391I 25 26 13 C406S 27 28 14 C406L 29 3015 P411L 31 32 16 C439S 33 34 17 C439L 35 36 18 C445S 37 38 19 R454F 3940 20 R464L 41 42 21 C482S 43 44 22 C482L 45 46 23 C507S 47 48 24C406S + C439S + C445S + 49 50 C482S + C507S 25 F378L 51 52 26 S495L 5354 27 G496L 55 56 28 M422S + M423L 57 58 29 V467S + S468L 59 60 30V467S + S468L + W470G 61 62 31 396-Leu-397 63 64 32 330-Leu-331 65 66 33456-Leu-457 67 68 34 475-Leu-476 69 70 35 367-Leu-368 71 72 36496-Leu-497 73 74

Each of the eAxmi205 proteins was expressed and purified as described inExample 2, although some proteins expressed at lower levels. In theseexperiments, eAxmi205 mutants #10 and #24 could not be purified insufficient quantities for further analysis; the other thirty-foureAxmi205 mutant proteins were successfully purified and were furtherevaluated in a preliminary SGF digestion analysis.

EXAMPLE 7 Preliminary Evaluation of eAxmi205 Mutants for SGF Digestion

The native eAxmi205 protein shows incomplete digestion in a standard SGFassay, with a persistent band at approximately 23 kDa. The 34 eAxmi205mutant proteins that were expressed and purified in Example 6 weresubjected to a single 10 minute time point (T10) evaluation for SGFdigestibility following the protocol described in Example 3. Mutantsthat showed no band at 23 kDa or lower at 10 minutes were deemed to have“passed” this preliminary T10 assessment (data not shown) and wereevaluated for bioactivity.

The following eAxmi205 mutants were deemed to “pass” the T10 SGFdigestion assessment: #5, #6, #21, #23, #28, #32, #33, #34, #35 and #36.Several eAxmi205 variants (#8, #29 and #30) were scored as “Fail/Pass”because while they showed much better digestion than native Axmi205, afaint band appeared to be present at 10 minutes. Because this was apreliminary screen, these Axmi205 variants were kept for furtherevaluation.

The other eAxmi205 mutants “failed” the preliminary T10 SGF digestionbecause one or more bands were present at 23 kDa or lower at 10 minutes,and were not evaluated further.

EXAMPLE 8 Control of Western Corn Rootworm by eAxmi205 Mutant Proteins

The eAxmi205 variants from Example 7 (#5, #6, #8, #21, #23, #28, #29,#30, #32, #33, #34, #35 and #36) that had acceptable SGF digestion at 10minutes were evaluated for activity in controlling WCRW in an artificialdiet bioassay following the protocol described in Example 4.

In the artificial diet bioassay, mortality was assessed at day 3 or 4and on day 6. Native Axmi205 (“Axmi205 wt”) was used as a positivecontrol, and buffer alone as a negative control. The data are shown inTables 2 to 4 below:

TABLE 2 % Mortality % Mortality WCR Day 4 WCR Day 6 Treatment Total DeadMort % Total Dead Mort % 1 Axmi205 wt @ 0.4 mg/mL (200 μg/mL FINAL) 12 758% 12 11 92% 2 Axmi205 mutant #5 @ 0.4 mg/mL (200 μg/mL FINAL) 12 3 25%12 7 58% 3 Axmi205 mutant #6 @ 0.4 mg/mL (200 μg/mL FINAL) 12 8 67% 12 975% 4 Axmi205 mutant #23 @ 0.4 mg/mL (200 μg/mL FINAL) 12 8 67% 12 1192% 5 Axmi205 mutant #28 @ 0.4 mg/mL (200 μg/mL FINAL) 12 9 75% 12 12100%  6 Axmi205 mutant #32 @ 0.4 mg/mL (200 μg/mL FINAL) 12 1  8% 12 1 8% 7 Axmi205 mutant #35 @ 0.4 mg/mL (200 μg/mL FINAL) 12 2 17% 12 3 25%8 buffer (1× PBS) 12 0  0% 12 0  0%

TABLE 3 % Mortality % Mortality WCR Day 4 WCR Day 6 Treatment Total DeadMort % Total Dead Mort % 1 Axmi205 wt @ 0.4 mg/mL (200 μg/mL FINAL) 12 867% 12 10  83% 2 Axmi205 mutant #8 @ 0.4 mg/mL (200 μg/mL FINAL) 12 1083% 12 12 100% 3 Axmi205 mutant #29 @ 0.4 mg/mL (200 μg/mL FINAL) 12 1 8% 12 1  8% 4 Axmi205 mutant #33 @ 0.4 mg/mL (200 μg/mL FINAL) 12 0  0%12 0  0% 5 Axmi205 mutant #34 @ 0.4 mg/mL (200 μg/mL FINAL) 12 10 83% 1212 100% 6 Axmi205 mutant #36 @ 0.4 mg/mL (200 μg/mL FINAL) 12 9 75% 1212 100% 7 buffer (1× PBS) 12 3 25% 12 4  33%

TABLE 4 % Mortality % Mortality WCR Day 3 WCR Day 6 Treatment Total DeadMort % Total Dead Mort % 1 Axmi205 wt @ 0.4 mg/mL (200 μg/mL FINAL) 12 650% 12 8 67% 2 Axmi205 mutant #21 @ 0.4 mg/mL (200 μg/mL FINAL) 12 5 42%12 9 75% 3 Axmi205 mutant #23 @ 0.4 mg/mL (200 μg/mL FINAL) 12 5 42% 129 75% 4 buffer (1× PBS) 12 0  0% 12 0  0%

EXAMPLE 9 Time Course Study of SGF Digestion of eAxmi205 Mutants

eAxmi205 mutants #5, #6, #21, #23, #28, #34 and #36 were subjected to amore detailed time course evaluation for digestion by SGF. Each mutantwas assessed for SGF digestion after 0, 1, 2, 5, 10, 30 and 60 minutes(i.e., T0, T1, T2, T5, T10, T30 and T60) as described in Example 5 byboth SDS-PAGE and Western blot analysis using a polyclonal rabbitantibody against native Axmi205. The results are summarized below.

eAxmi205 #5:

On SDS-PAGE, the band corresponding to full-length Axmi205-5 at timezero (T0) was no longer visible after incubation in SGF for one minute.After 1 minute, 56 kDa, 23 kDa, 4 kDa and 3 kDa fragments appeared. Avery faint 53 kDa band is seen at 5 minutes, and disappeared by 10minutes. The 23 kDa fragment is not seen in the T30 sample. 3 and 4 kDafragments were still visible after 60 minutes incubation in SGF.

Western blot analysis confirmed the results of SDS-PAGE. Full-lengthAxmi205-5 was no longer visible at 1 minute. The 56 kDa band is nolonger visible in the T10 sample, and the 23 kDa fragment was no longervisible in the T30 sample. No other immunoreactive fragments weredetected.

eAxm 1205 #6:

The band corresponding to full-length Axmi205-6 at time zero (T0) was nolonger visible after incubation in SGF for 1 minute. At the T1 timepoint, 56 kDa, 23 kDa, 4 kDa and 3 kDa fragments appeared. A very faint53 kDa band is seen at T5, and disappears by T10. A very faint 23 kDaband is observed at T10, and disappears in the T30 sample. 3 and 4 kDafragments were still visible after 60 minutes incubation in SGF.

Western blot analysis confirmed the results of SDS-PAGE. Full-lengthAxmi205-6 was no longer visible at 1 minute. The 56 kDa band is nolonger visible in the T10 sample, and a very faint 23 kDa fragment wasvisible in the T30 sample. No other immunoreactive fragments weredetected.

eAxm 1205 #21:

On SDS-PAGE, the band corresponding to full-length Axmi205-21 at timezero (T0) was no longer visible after incubation in SGF for one minute.After 1 minute of incubation, 56 kDa, 23 kDa, 4 kDa and 3 kDa fragmentsappeared. 56 kDa and 23 kDa fragments were no longer visible after 5 and10 minutes of incubation in SGF, respectively. 4 kDa and 3 kDa fragmentsdiminished in intensity over time, however, were still visible after 60minutes incubation in SGF.

Western blot analysis confirmed the results of SDS-PAGE. Full-lengthAxmi205-21 was no longer visible after incubation in SGF for one minute.Very faint bands corresponding to 56 kDa and 23 kDa fragments werevisible after 5 minutes of incubation in SGF, which disappeared after 10min. No other immunoreactive fragments were detected.

eAxmi205 #23:

On SDS-PAGE, the band corresponding to full-length Axmi205-23 at timezero (T0) was no longer visible after incubation in SGF for one minute.After 1 minute of incubation, 56 kDa, 23 kDa, 4 kDa and 3 kDa fragmentsappeared. Both 56 kDa and 23 kDa fragments were no longer visible after5 minutes of incubation in SGF. 4 kDa and 3 kDa fragments diminished inintensity over time, however, they were still visible after 60 minutesincubation in SGF.

Western blot analysis confirmed the results of SDS-PAGE. Full-lengthAxmi205-23 was no longer visible after incubation in SGF for one minute.56 kDa and 23 kDa fragments were not visible after 5 and 2 minutes ofincubation in SGF, respectively. No other immunoreactive fragments weredetected.

eAxm 1205 #28:

On SDS-PAGE, the band corresponding to full-length Axmi205-28 at timezero (T0) was no longer visible after incubation in SGF for one minute.At 1 minute, 56 kDa, 23 kDa, 4 kDa and 3 kDa fragments appeared. Both 53and 23 kDa fragment bands were not observed at 5 minutes. 3 and 4 kDafragments were still visible after 60 minutes incubation in SGF.

Western blot analysis confirmed the results of SDS-PAGE. Full-lengthAxmi205-28 was no longer visible at one minute. The 56 kDa fragment wasno longer visible at 5 minutes. A very faint 23 kDa band was stillvisible at 2 minutes, but not observed at 5 minutes. No otherimmunoreactive fragments were detected.

eAxmi205 #34:

On SDS-PAGE, the band corresponding to full-length Axmi205-34 at timezero (T0) was no longer visible after incubation in SGF for one minute.At one minute, 4 kDa and 3 kDa fragments appeared and were still visibleafter 60 minutes incubation in SGF. No other significant Coomassiestained bands were observed on the gel.

Western blot analysis confirmed the results of SDS-PAGE. Full-lengthAxmi205-34 was no longer visible after one minute of SGF digestion. Noother immunoreactive fragments were detected.

eAxmi205 #36:

On SDS-PAGE, the band corresponding to full-length Axmi205-36 (˜59 kDa)at time zero (T0) was no longer visible after incubation in SGF for oneminute (T1). At one minute, a smaller fragment (˜56 kDa) appeared, butwas no longer observed at T2. Fragments at 4 kDa and 3 kDa appeared atT1, and were still visible after 60 minutes incubation in SGF. No othersignificant Coomassie stained bands were observed on the gel.

Western blot analysis confirmed the results of SDS-PAGE. Full-lengthAxmi205-34 was no longer visible after one minute of SGF digestion. Noother immunoreactive fragments were detected.

EXAMPLE 10 Summary of Results from Examples 6 to 9

Table 5 below provides a summary of the results discussed Examples 6 to9 above. FIGS. 2-10 show SGF assay results.

TABLE 5 Protein WCRW SGF Time # eAxmi205 Mutant Expression Purified? SGFT10 Activity? Course 1 K328Y ++++ Yes Fail 2 K328L ++++ Yes Fail 3 K328F++++ Yes Fail 4 Y404F ++++ Yes Fail 5 K402F ++++ Yes Pass Yes Yes 6K402N ++++ Yes Pass Yes Yes 7 K402L +++ Yes Fail 8 Y404F + K402L +++ YesFail/Pass Yes Not Tested 9 R416L +++ Yes Fail 10 P386L + No N/A 11 R391L++++ Yes Fail 12 R391I ++++ Yes Fail 13 C406S ++++ Yes Fail 14 C406L +++Yes Fail 15 P411L ++ Yes Fail 16 C439S + Yes Fail 17 C439L ++ Yes Fail18 C445S ++++ Yes Fail 19 R454F ++++ Yes Fail 20 R464L ++++ Yes Fail 21C482S ++++ Yes Pass Yes Yes 22 C482L ++++ Yes Fail 23 C507S ++++ YesPass Yes Yes 24 C406S + C439S + + No N/A C445S + C482S + C507S 25 F378L+++ Yes Fail 26 S495L +++ Yes Fail 27 G496L ++++ Yes Fail 28 M422S +M423L ++++ Yes Pass Yes Yes 29 V467S + S468L ++++ Yes Fail/Pass NO 30V467S + S468L + +++ Yes Fail/Pass W470G 31 396-Leu-397 +++ Yes Fail 32330-Leu-331 +++ Yes Pass NO 33 456-Leu-457 ++++ Yes Pass NO 34475-Leu-476 ++++ Yes Pass Yes Yes 35 367-Leu-368 +++ Yes Pass NO 36496-Leu-497 ++++ Yes Pass Yes Yes

EXAMPLE 11 Dose-Response for WCRW Control Activity

A dose-response study for control of WCRW in an artificial diet bioassaywas carried out for native Axmi205 (“Axmi205 WT”) and eAxmi205 mutants#23, #28, and #34 using essentially the protocol described in Example 4.Mortality was assessed at days 1, 4 and 6.

The study was done in two replicates of 15 insects for each treatment.The results are shown in Table 6:

TABLE 6 % Mortality WCRW Day 1 Day 4 Day 6 Treatment Total Dead Mort %Dead Mort % Dead Mort % Replicate 1. Axmi205 WT @ 0.6 mg/mL (300 μg/mLFINAL) 15 0 0% 11 73% 15 100%  Axmi205 WT @ 0.4 mg/mL (200 μg/mL FINAL)15 0 0% 13 87% 15 100%  Axmi205 WT @ 0.2 mg/mL (100 μg/mL FINAL) 15 0 0%12 80% 14 93% Axmi205 WT @ 0.1 mg/mL (50 μg/mL FINAL) 15 0 0% 6 40% 1493% Axmi205 WT @ 0.05 mg/mL (25 μg/mL FINAL) 15 0 0% 3 20% 8 53% Axmi205WT Buffer (Neg) 15 0 0% 3 20% 4 27% Axmi205 Mutant #23 @ 0.6 mg/mL (300μg/mL FINAL) 15 0 0% 12 80% 15 100%  Axmi205 Mutant #23 @ 0.4 mg/mL (200μg/mL FINAL) 15 0 0% 12 80% 15 100%  Axmi205 Mutant #23 @ 0.2 mg/mL (100μg/mL FINAL) 15 0 0% 10 67% 13 87% Axmi205 Mutant #23 @ 0.1 mg/mL (50μg/mL FINAL) 15 0 0% 5 33% 10 67% Axmi205 Mutant #23 @ 0.05 mg/mL (25μg/mL FINAL) 15 0 0% 3 20% 8 53% Axmi205 Mutant #23 Buffer (Neg) 15 0 0%1  6% 3 20% Axmi205 Mutant #28 @ 0.6 mg/mL (300 μg/mL FINAL) 15 0 0% 15100%  15 100%  Axmi205 Mutant #28 @ 0.4 mg/mL (200 μg/mL FINAL) 15 1 7%14 93% 15 100%  Axmi205 Mutant #28 @ 0.2 mg/mL (100 μg/mL FINAL) 15 0 0%9 60% 14 93% Axmi205 Mutant #28 @ 0.1 mg/mL (50 μg/mL FINAL) 15 0 0% 853% 14 93% Axmi205 Mutant #28 @ 0.05 mg/mL (25 μg/mL FINAL) 15 0 0% 1067% 15 100%  Axmi205 Mutant #28 Buffer (Neg) 15 0 0% 1  6% 5 33% Axmi205Mutant #34 @ 0.6 mg/mL (300 μg/mL FINAL) 15 0 0% 14 93% 15 100%  Axmi205Mutant #34 @ 0.4 mg/mL (200 μg/mL FINAL) 15 0 0% 11 73% 14 92% Axmi205Mutant #34 @ 0.2 mg/mL (100 vg/mL FINAL) 15 0 0% 14 93% 15 100%  Axmi205Mutant #34 @ 0.1 mg/mL (50 μg/mL FINAL) 15 0 0% 12 80% 13 87% Axmi205Mutant #34 @ 0.05 mg/mL (25 μg/mL FINAL) 15 0 0% 8 53% 9 60% Axmi205Mutant #34 Buffer (Neg) 15 0 0% 0 0 2 17% Replicate 2. Axmi205 WT @ 0.6mg/mL (300 μg/mL FINAL) 15 0 0% 13 87% 15 100%  Axmi205 WT @ 0.4 mg/mL(200 μg/mL FINAL) 15 0 0% 11 73% 15 100%  Axmi205 WT @ 0.2 mg/mL (100μg/mL FINAL) 15 0 0% 12 80% 14 93% Axmi205 WT @ 0.1 mg/mL (50 μg/mLFINAL) 15 0 0% 7 47% 13 87% Axmi205 WT @ 0.05 mg/mL (25 μg/mL FINAL) 150 0% 6 40% 14 93% Axmi205 WT Buffer (Neg) 15 0 0% 3 20% 10 67% Axmi205Mutant #23 @ 0.6 mg/mL (300 μg/mL FINAL) 15 0 0% 15 100%  15 100% Axmi205 Mutant #23 @ 0.4 mg/mL (200 μg/mL FINAL) 15 0 0% 11 73% 14 92%Axmi205 Mutant #23 @ 0.2 mg/mL (100 μg/mL FINAL) 15 0 0% 12 80% 15 100% Axmi205 Mutant #23 @ 0.1 mg/mL (50 μg/mL FINAL) 15 0 0% 3 20% 13 87%Axmi205 Mutant #23 @ 0.05 mg/mL (25 μg/mL FINAL) 15 0 0% 3 20% 8 53%Axmi205 Mutant #23 Buffer (Neg) 15 0 0% 1  7% 2 17% Axmi205 Mutant #28 @0.6 mg/mL (300 μg/mL FINAL) 15 0 0% 13 87% 14 93% Axmi205 Mutant #28 @0.4 mg/mL (200 μg/mL FINAL) 15 0 0% 14 93% 15 100%  Axmi205 Mutant #28 @0.2 mg/mL (100 μg/mL FINAL) 15 0 0% 10 67% 14 93% Axmi205 Mutant #28 @0.1 mg/mL (50 μg/mL FINAL) 15 0 0% 6 40% 13 87% Axmi205 Mutant #28 @0.05 mg/mL (25 μg/mL FINAL) 15 0 0% 4 27% 11 73% Axmi205 Mutant #28Buffer (Neg) 15 0 0% 1  7% 9 60% Axmi205 Mutant #34 @ 0.6 mg/mL (300μg/mL FINAL) 15 0 0% 12 80% 15 100%  Axmi205 Mutant #34 @ 0.4 mg/mL (200μg/mL FINAL) 15 0 0% 10 67% 15 100%  Axmi205 Mutant #34 @ 0.2 mg/mL (100μg/mL FINAL) 15 1 8% 13 87% 15 100%  Axmi205 Mutant #34 @ 0.1 mg/mL (50μg/mL FINAL) 15 0 0% 10 67% 12 80% Axmi205 Mutant #34 @ 0.05 mg/mL (25μg/mL FINAL) 15 0 0% 8 53% 11 73% Axmi205 Mutant #34 Buffer (Neg) 15 00% 0  0% 2 17%

Surprisingly, all 3 of the eAxmi205 mutants tested were essentially asactive as native Axmi205 in controlling WCRW, while having a moredesirable SGF digestion profile.

EXAMPLE 12 Additional eAxmi205 Variants

Based on the information presented in Table 5 above, additional eAxmi205mutants with an improved SGF digestion profile are readily generated byone skilled in the art. Mutants can add a new pepsin cleavage site,reduce disulfide bond formation and/or otherwise modify and “open up”the tertiary structure of the protein to increase enzyme access.

For example, eAxmi205 mutant #21 has a C482S mutation (with respect tothe reference amino acid sequence of native Axmi205 of SEQ ID NO: 1).Alternatively, any other amino acid, naturally occurring or synthetic,is substituted at position 482. For example, the amino acid substitutionat position 482 is a substitution of an amino acid with an aliphatichydrophobic side chain (e.g., A, I, L or V), an amino acid with anaromatic hydrophobic side chain (e.g., F, W or Y), an amino acid with apolar neutral side chain (e.g., N, Q, M or T), an amino acid with anacidic side chain (e.g., D or E), an amino acid with a basic side chain(e.g., R, H or K), a G, or a P. In embodiments, the substitution resultsin a new pepsin cleavage site directly before and/or directly after thesubstitution at position 482 (i.e., between residues 481 and 482 and/orbetween residues 482 and 483). Additional mutants, eAxmi205 mutant #21Fand #21D (C482F, C482D) were generated and the data for these mutationsare included in Table 7.

As another non-limiting example, eAxmi205 mutant #23 has a C507Smutation (with respect to reference amino acid sequence of nativeAxmi205 of SEQ ID NO: 1). Alternatively, any other amino acid, naturallyoccurring or synthetic, is substituted at position 507. For example, theamino acid substitution at position 507 is a substitution of an aminoacid with an aliphatic hydrophobic side chain (e.g., A, I, L or V), anamino acid with an aromatic hydrophobic side chain (e.g., F, W or Y), anamino acid with a polar neutral side chain (e.g., N, Q, M or T), anamino acid with an acidic side chain (e.g., D or E), an amino acid witha basic side chain (e.g., R, H or K), a G, or a P. In embodiments, thesubstitution results in a new pepsin cleavage site directly beforeand/or directly after the substitution at position 507 (i.e., betweenresidues 506 and 507 and/or between residues 507 and 508). Additionalmutants, eAxmi205 mutant #23L, #23A, #23F, #23D, and #23R (C507L, C507A,C507F, C507D, C507R) were generated and the data for these mutations areincluded in Table 7.

As a further illustration, eAxmi205 mutant #28 has two substitutionmutations: M422S and M423L (with respect to the reference amino acidsequence of native Axmi205 of SEQ ID NO: 1). Alternatively, an eAxmi205variant with only one of the substitutions is made, i.e., M422S orM423L. As a further alternative, any other amino acid, naturallyoccurring or synthetic, is substituted at position 422 and/or position423. For example, the amino acid substitution at position 422 and/orposition 423 is a substitution of an amino acid with an aliphatichydrophobic side chain (e.g., A, I, L or V), an amino acid with anaromatic hydrophobic side chain (e.g., F, W or Y), an amino acid with apolar neutral side chain (e.g., N, C, Q, S or T), an amino acid with anacidic side chain (e.g., D or E), an amino acid with a basic side chain(e.g., R, H or K), a G, or a P. In embodiments, the substitution(s)results in a new pepsin cleavage site directly before residue 422,between residues 422 and 423 and/or directly following residue 423(i.e., between residues 421 and 422, between residues 422 and 423 and/orbetween residues 423 and 424). In embodiments, the substitution is not acysteine (C) at position 422 and/or position 423. Additional mutants,eAxmi205 mutant #28TF, #28DE, #28KR, #28SE and #28KF (M422T+M423F,M422D+M423E, M422K+M423R, M422S+M423E, M422K+M423F) were generated andthe data for these mutations are included in Table 7.

eAxmi205 mutant #34 has a leucine (L) insertion between amino acids 475and 476 (with respect to the reference amino acid sequence of nativeAxmi205 of SEQ ID NO: 1). Alternatively, any other amino acid, naturallyoccurring or synthetic, is inserted between the amino acid residues atpositions 475 and 476. For example, the amino acid insertion betweenpositions 475 and 476 is an insertion of an amino acid with an aliphatichydrophobic side chain (e.g., A, I or V), an amino acid with an aromatichydrophobic side chain (e.g., F, W or Y), an amino acid with a polarneutral side chain (e.g., N, C, Q, M, Q, S or T), an amino acid with anacidic side chain (e.g., D or E), an amino acid with a basic side chain(e.g., R, H or K), a G, or a P. In embodiments, the insertion results ina new pepsin cleavage site directly before and/or directly after theinsertion (i.e., between residue 475 and the inserted amino acid and/orbetween the inserted amino acid and residue 476). In embodiments, theinserted amino acid is not a cysteine (C). Additional mutants, eAxmi205mutant #34F, #34D and #34R (475-Phe-476, 475-Asp-476, 475-Arg-476) weregenerated and the data for these mutations are included in Table 7.

eAxmi205 mutant #36 has a leucine (L) insertion between amino acids 496and 497 (with respect to the reference amino acid sequence of nativeAxmi205 of SEQ ID NO: 1). Alternatively, any other amino acid, naturallyoccurring or synthetic, is inserted between the amino acid residues atpositions 496 and 497. For example, the amino acid insertion betweenpositions 496 and 497 is an insertion of an amino acid with an aliphatichydrophobic side chain (e.g., A, I or V), an amino acid with an aromatichydrophobic side chain (e.g., F, W or Y), an amino acid with a polarneutral side chain (e.g., N, C, Q, M, Q, S or T), an amino acid with anacidic side chain (e.g., D or E), an amino acid with a basic side chain(e.g., R, H or K), a G, or a P. In embodiments, the insertion results ina new pepsin cleavage site directly before and/or directly after theinsertion (i.e., between residue 496 and the inserted amino acid and/orbetween the inserted amino acid and residue 497. In embodiments, theinserted amino acid is not a cysteine (C). Additional mutants, eAxmi205mutant #36F and #36R (496-Phe-497, 496-Arg-497) were generated and thedata for these mutations are included in Table 7.

eAxmi205 mutants #5 and #6 have K402F and K402N mutations, respectively(with respect to the reference amino acid sequence of native Axmi205 ofSEQ ID NO: 1). Alternatively, any other amino acid, naturally occurringor synthetic, is substituted at position 402. For example, the aminoacid substitution at position 402 is a substitution of an amino acidwith an aliphatic hydrophobic side chain (e.g., A, I or V), an aminoacid with an aromatic hydrophobic side chain (e.g., W or Y), an aminoacid with a polar neutral side chain (e.g., C, Q, M, Q, S or T), anamino acid with an acidic side chain (e.g., D or E), an amino acid witha basic side chain (e.g., R or H), a G, or a P. In embodiments, thesubstitution results in a new pepsin cleavage site directly before ordirectly after the substitution at position 402 (i.e., between residues401 and 402 or between residues 402 and 403). An additional mutant,eAxmi205 mutant #5D (K402D) was generated and the data for this mutationis included in Table 7.

eAxmi205 mutant #8 has two substitution mutations: K402L and Y404F (withrespect to the reference amino acid sequence of native Axmi205 of SEQ IDNO: 1). Alternatively, any other amino acid, naturally occurring orsynthetic, is substituted at position 402 and/or 404. For example, theamino acid substitution at position 402 and/or position 404 is asubstitution of an amino acid with an aliphatic hydrophobic side chain(e.g., A, I, L or V), an amino acid with an aromatic hydrophobic sidechain (e.g., F, W or Y), an amino acid with a polar neutral side chain(e.g., N, C, M, Q, S or T), an amino acid with an acidic side chain(e.g., D or E), an amino acid with a basic side chain (e.g., R, H or K),a G, or a P. In embodiments, the substitution(s) results in a new pepsincleavage site directly before residue 402 and/or directly followingresidue 404 (i.e., between residues 401 and 402 and/or between residues404 and 405). In embodiments, the substitution is not a cysteine (C) atposition 402 and/or position 404.

As further alternatives, any combination of the mutations described inTable 5 or in this example are combined to generate additional eAxmi205variants, for example, combination of the mutations at positions 402 and404.

The additional eAxmi205 variants can be assessed for SGF digestion asdescribed in Examples 3 and/or 5 and for insecticidal activity byartificial diet bioassay as described in Example 4 and/or by expressionin plants (described below).

As further alternatives, additional or alternative insertions can bemade in long regions of the protein without a pepsin cleavage site.Based upon the SGF results of mutants #34 and #36 and their variants,additional mutants were created in the stretch of amino acids betweenpositions 469 and 483 of SEQ ID NO: 1 and between positions 483 and 501of SEQ ID NO:1. Additional mutants, eAxmi205 mutant #37F and #37L(471-Phe-472, 471-Leu-472), #38F and #38L (479-Phe-480, 479-Leu-480),and #39F and #39L (489-Phe-490, 489-Leu-490) were generated and the datafor these mutations are included in Table 7.

Table 7 shows data for eAxmi205 mutants #5, #21, #23, #28, #34, #36,#37, #38, #39 and their variants as listed above during this example.These mutants were assessed for SGF digestion as described in Examples 3and/or 5. From the data it is clear that in all cases, the additionalmutants at these positions improved the digestion of the modifiedAxmi205 toxin. These modified Axmi205 toxins showed enhanceddigestibility by a mammalian protease such that there was a lesseramount of fragments above 4 kDa remaining as compared with an Axmi205toxin that did not comprise the modification when tested under the sameconditions. The four mutants which did not pass the T10 test stillshould enhanced digestibility after 60 minutes. FIGS. 11-15 show SGFassay results.

T10 Improved v. WT # Mutant Position Test Axmi205 at T10  5 K402F PassYes  5D K402D Fail Yes 21 C482S Pass Yes 21F C482F Fail Yes 21D C482DFail Yes 23 C507S Pass Yes 23L C507L Fail Yes 23A C507A Pass Yes 23FC507F Pass Yes 23D C507D Pass Yes 23R C507R Pass Yes 28 M422S + M423LPass Yes 28TF M422T + M423F Pass Yes 28DE M422D + M423E Pass Yes 28KRM422K + M423R Pass Yes 28SE M422S + M423E Pass Yes 28KF M422K + M423FPass Yes 34 475-Leu-476 Pass Yes 34F 475-Phe-476 Pass Yes 34D475-Asp-476 Pass Yes 34R 475-Arg-476 Pass Yes 36 496-Leu-497 Pass Yes36D 496-Asp-497 Pass Yes 36F 496-Phe-497 Pass Yes 36R 496-Arg-497 PassYes 37F 471-Phe-472 Pass Yes 37L 471-Leu-472 Pass Yes 38F 479-Phe-480Pass Yes 38L 479-Leu-480 Pass Yes 39F 489-Phe-490 Pass Yes 39L489-Leu-490 Pass Yes

Any of the above mutants can be combined with one another in an effortto further improve digestibility. A key finding of these results is thatcertain stretches of amino acids, the stretch of amino acids betweenpositions 469 and 483 of SEQ ID NO: 1 and between positions 483 and 501of SEQ ID NO:1, appear to have a strong effect on pepsin cleavage. Theinsertion of an amino acid at different points in these two stretchesled to significantly increased digestibility, indicating that they arekey for pepsin cleavage.

EXAMPLE 13 Expression and Activity of eAxmi205 Variants in MonocotPlants

A binary vector construct suitable for Agrobacterium-mediatedtransformation is produced. The binary vector comprises a maizeoptimized eAxmi205 coding sequence operably linked at the 5′ end to apromoter suitable for driving expression in maize plants and operablylinked at the 3′ end to a terminator sequence. Examples of suitablemaize codon optimized sequences include SEQ ID NO: 75 (eAxmi205 #23),SEQ ID NO: 76 (eAxmi205 #28), and SEQ ID NO: 77 (eAxmi205 #34).

The binary vector is transformed into Agrobacterium tumefaciens usingstandard molecular biology techniques known to those skilled in the art.To prepare the Agrobacteria for transformation, cells are cultured inliquid YPC media at 28° C. and 220 rpm overnight. Agrobacteriumtransformation of immature maize embryos is performed essentially asdescribed in Negrotto et al., 2000, (Plant Cell Reports 19: 798-803);however, various protocols known in the art may be used.

Following transformation, selection, and regeneration, maize plants areassayed for the presence of the eAxmi204 coding sequence using TaqMan®analysis. Plants are also tested for the presence of the vectorbackbone. Plants negative for the vector backbone and comprising onecopy of the transgene from the binary vector construct are transferredto the greenhouse and tested for resistance to WCRW damage.

EXAMPLE 14 Expression and Activity of eAxmi205 Variants in Dicot Plants

Transformation of soybean to produce transgenic soybean plants isaccomplished using mature seed targets of variety Williams 82 via A.tumefaciens-mediated transformation using explant materials and mediarecipes essentially as described in Hwang et al. (WO 08/112044) and Queet al. (WO 08/112267); however, various other protocols can also beused.

Following transformation, selection and regeneration, soybean plants areassayed for the presence of the eAxmi204 coding sequence using TaqMan®analysis. Plants are also tested for the presence of the vectorbackbone. Plants negative for the vector backbone and comprising onecopy of the transgene from the binary vector construct are transferredto the greenhouse and tested for resistance to damage by Bean leafbeetle (Cerotoma trifurcate).

EXAMPLE 15 Activity Against Resistant Corn Rootworm

The disclosed Axmi205 variants are useful to control coleopteran insectpests that have developed resistance against another coleopteran insectcontrol agent (e.g., a protein toxin, chemical, microbial and/or RNAicontrol agent). The Axmi205 variants can also be combined with one ormore other coleopteran insect control agents to delay or prevent thedevelopment of resistance in the coleopteran insect population.

The Axmi205 variants (e.g., #5, #6, #21, #21, #23, #28, #34 and/or #36)are tested in diet bioassay or in planta assay for activity against acorn rootworm (e.g., WCRW) colony that is resistant to a coleopteraninsect control agent, such as mCry3A (e.g., maize event MIR604;Syngenta), eCry3.1Ab (e.g., maize event 5307; Syngenta), Cry3Bb1 (e.g.,maize event MON88017; Monsanto), Cry34/35Ab1 (e.g., maize eventDAS-59122, Dow AgroSciences), or RNAi traits, such as DvSnf7 dsRNA(e.g., maize event MON87411; Monsanto).

In one experiment, Axmi205 mutant #34 (SEQ ID NO: 69) was evaluated inan artificial diet bioassay against a WCRW colony with resistanceagainst the toxin eCry3.1Ab, essentially as described above in Example4. The results of 2 replicates are shown below in Table 8. Results forthe additional mutants described in Example 12 are shown in Table 9.

TABLE 8 % Mortality % Mortality WCR-r Day 4 WCR-r Day 6 Total Dead Mort% Total Dead Mort % REPLICATE 1 Axmi205 Mutant #34 @ 0.4 mg/mL (200μg/mL FINAL) 12 4 33% 12 4 33% Axmi205 Mutant #34 @ 0.2 mg/mL (100 μg/mLFINAL) 12 2 17% 12 8 67% Axmi205 Mutant #34 @ 0.1 mg/mL (50 μg/mL FINAL)12 0  0% 12 3 25% Axmi205 Mutant #34 @ 0.05 mg/mL (25 μg/mL FINAL) 12 0 0% 12 5 42% Axmi205 Mutant #34 @ 0.025 mg/mL (12.5 μg/mL FINAL) 12 1 8% 12 3 25% Axmi205 Mutant #34 buffer (Negative control) (1× PBS) 12 1 8% 12 1  8% REPLICATE 2 Axmi205 Mutant #34 @ 0.4 mg/mL (200 μg/mLFINAL) 12 8 67% 12 11 92% Axmi205 Mutant #34 @ 0.2 mg/mL (100 μg/mLFINAL) 12 10 83% 12 12 100%  Axmi205 Mutant #34 @ 0.1 mg/mL (50 μg/mLFINAL) 12 3 25% 12 4 33% Axmi205 Mutant #34 @ 0.05 mg/mL (25 μg/mLFINAL) 12 4 33% 12 7 58% Axmi205 Mutant #34 @ 0.025 mg/mL (12.5 μg/mLFINAL) 12 0  0% 12 3 25% Axmi205 Mutant #34 buffer (Negative control)(1× PBS) 12 2 17% 12 2 17%

TABLE 9 % Mortality WCR-s Day 3 Day 6 Treatment Total Dead Mort %Remarks Dead Mort % Remarks 1 Axmi205 WT @ 0.4 mg/mL (200 ug/mL 12 1083% 1s, 1m 12 100%  FINAL) 2 Axmi205 21D @ 0.4 mg/mL (200 ug/mL 12 3 25%mb/b 9 75% b FINAL) 3 Axmi205 28SE @ 0.4 mg/mL (200 ug/mL 12 6 50% mb/b10 83% mb FINAL) 4 Axmi205 28KF @ 0.4 mg/mL (200 ug/mL 12 0  0% b 0  0%b FINAL) 5 Axmi205 36F @ 0.4 mg/mL (200 ug/mL 12 3 25% mb/b 7 58% bFINAL) 6 Axmi205 36R @ 0.4 mg/mL (200 ug/mL 12 5 42% mb/b 8 67% 1m, 3bFINAL) 7 Axmi205 37F @ 0.4 mg/mL (200 ug/mL 12 5 42% m 10 83% mb FINAL)8 Axmi205 37L @ 0.4 mg/mL (200 ug/mL 12 3 25% mb/b 6 50% b FINAL) 9Axmi205 38F @ 0.4 mg/mL (200 ug/mL 12 5 42% mb/b 9 75% b FINAL) 10Axmi205 38L @ 0.4 mg/mL (200 ug/mL 12 4 33% mb/b 7 58% mb/b FINAL) 11Axmi205 39F @ 0.4 mg/mL (200 ug/mL 12 8 67% sm 11 92% m FINAL) 12Axmi205 39L @ 0.4 mg/mL (200 ug/mL 12 2 17% mb 10 83% b FINAL) 13 Buffer(1× PBS) 12 2 17% b 2 17% b

EXAMPLE 16 eAxmi205 in Combination with an Insecticidal Interfering RNA

An eAxmi205 variant as described herein is expressed and purified asdescribed in Example 2. dsRNA against an essential target gene in WCRWis prepared. In a non-limiting example, the dsRNA may target a geneencoding vacuolar ATP synthase, beta-tubulin, 26S proteosome subunit p28protein, EF1α 48D, troponin I, tetraspanin, gamma-coatomer,beta-coatomer, and/or juvenile hormone epoxide hydrolase (U.S.Provisional Application Nos. 62/371,259, 62/371,261, and 62/371,262;U.S. Pat. No. 7,812,219; each herein incorporated by reference). ThedsRNA and purified protein are tested for efficacy against WCRW in adiet-incorporation assay, performed essentially as described in Example4.

EXAMPLE 17 Genome Editing in Plant Cells In Situ to Generate ModifiedNucleic Acid Sequences Encoding eAxmi205 Variants

The following Example illustrates the use of genome editing of a plantcell genome in situ to incorporate the mutations described herein(including but not limited to the mutations described in Table 5 andExample 12) into a coding sequence for the native Axmi205 protein (SEQID NO: 1) or into a coding sequence for an already modified Axmi205protein.

Targeted genome modification, also known as genome editing, is usefulfor introducing mutations in specific DNA sequences. These genomeediting technologies, which include zinc finger nucleases (ZNFs),transcription activator-like effector nucleases (TALENS), meganucleasesand clustered regularly interspaced short palindromic repeats (CRISPR)have been successfully applied to over 50 different organisms includingcrop plants. See, e.g., Belhaj, K., et al., Plant Methods 9, 39 (2013);Jiang, W., et al., Nucleic Acids Res, 41, e188 (2013)). The CRISPR/Cassystem for genome editing is based on transient expression of Cas9nuclease and an engineered single guide RNA (sgRNA) that specifies thetargeted polynucleotide sequence.

Cas9 is a large monomeric DNA nuclease guided to a DNA target sequencewith the aid of a complex of two 20-nucleotide (nt) non-coding RNAs:CRIPSR RNA (crRNA) and trans-activating crRNA (tracrRNA), which arefunctionally available as single synthetic RNA chimera. The Cas9 proteincontains two nuclease domains homologous to RuvC and HNH nucleases. TheHNH nuclease domain cleaves the complementary DNA strand, whereas theRuvC-like domain cleaves the non-complementary strand and, as a result,a blunt cut is introduced in the target DNA.

When the Cas9 and the sgRNA are transiently expressed in living maizecells, double strand breaks (DSBs) in the specific targeted DNA iscreated in the transgenic maize cell. Mutation at the break site isintroduced through the non-homologous end joining and homology-directedDNA repair pathways.

Specific mutations, for example the eAxmi205 mutations described inTable 5 and Example 12 above, are introduced into a coding sequence forthe native Axmi205 (SEQ ID NO: 1) or a modified Axmi205, through the useof recombinant plasmids expressing the Cas9 nuclease and the sgRNAtarget that is maize codon optimized for the axmi205 or modified axmi205sequence in the transgenic maize. Implementation of the method is by anagroinfiltration method with Agrobacterium tumefaciens carrying thebinary plasmid harboring the specified target sequence of interest.After the sgRNA binds to the target axmi205 or modified axmi205 codingsequence, the Cas9 nuclease makes specific cuts into the coding sequenceand introduces the desired mutation(s) during DNA repair. Thus, a nowmutated axmi205 coding sequence will encode an eAxmi205 variant protein,such as the variants described in Table 5, for example, where a mutationat position 507 replaces cysteine (C) with serine (S), or where amutation at position 422 replaces methionine (M) with a serine (S)combined with a mutation at position 423 that replaces a methionine (M)with a leucine (L), or where a leucine (L) is inserted between the aminoacids at positions 475 and 476 and/or between the amino acids atpositions 496 and 497.

Plant cells comprising the genome edited eAxmi205 coding sequences arescreened by PCR and sequencing. Calli that harbor genome editedmutations in the axmi205 or modified axmi205 coding sequences areinduced to regenerate plants for phenotype evaluation for insecticidalactivity of the expressed eAxmi205 protein against WCRW, Northern CornRootworm (Diabrotica barberi), Southern Corn Rootworm (Diabroticaundecimpunctata howardi) and/or Mexican Corn Rootworm (Diabroticavirgifera zeae).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed:
 1. A modified Axmi205 toxin, wherein saidmodified Axmi205 toxin comprises an amino acid sequence having at least99% identity to the polypeptide of SEQ ID NO: 1 and comprises asubstitution of a serine, leucine, alanine, phenylalanine, aspartic acidor arginine at position C507 in the polypeptide of SEQ ID NO:1, andwherein the substitution results in enhanced digestion of the modifiedAxmi205 toxin by pepsin as compared with the Axmi205 toxin that does notcomprise the substitution.
 2. The modified Axmi205 toxin of claim 1,wherein said toxin is active against an insect pest selected from thegroup consisting of Western corn rootworm (Diabrotica virgiferavirgifera), Northern Corn Rootworm (Diabrotica barberi), Southern CornRootworm (Diabrotica undecimpunctata howardi) and Mexican Corn Rootworm(Diabrotica virgifera zeae).
 3. The modified Axmi205 toxin of claim 1,wherein the modified Axmi205 toxin comprises an amino acid substitutionof C5075 in the polypeptide of SEQ ID NO:1 or the corresponding cysteineresidue in the amino acid sequence having at least 99% identity to SEQID NO:1.
 4. The modified Axmi205 toxin of claim 1, wherein the modifiedAxmi205 toxin comprises the amino acid sequence of SEQ ID NO:
 47. 5. Apolynucleotide comprising a nucleotide sequence encoding the modifiedAxmi205 toxin according to claim
 1. 6. The polynucleotide according toclaim 5, wherein the polynucleotide comprises the nucleotide sequence ofSEQ ID NO: 48 or
 75. 7. A nucleic acid molecule comprising thepolynucleotide according to claim 5 operably associated with aheterologous promoter.
 8. A vector comprising the nucleic acid moleculeaccording to claim
 7. 9. A transgenic plant comprising the nucleic acidmolecule of claim
 7. 10. The transgenic plant according to claim 9,wherein the transgenic plant is a maize plant.
 11. A transgenic seed ofthe transgenic plant according to claim 10, wherein the seed comprisesthe nucleic acid molecule.
 12. A transgenic seed of the transgenic plantaccording to claim 11, wherein the seed comprises the nucleic acidmolecule.
 13. A method of producing a transgenic plant with increasedresistance to a coleopteran insect pest, the method comprisingintroducing into a plant the polynucleotide of claim 5, wherein themodified Axmi205 toxin is expressed in the plant, thereby producing atransgenic plant with increased resistance to a coleopteran insect pest.14. The method according to claim 13, wherein the introducing stepcomprises: i. transforming a plant cell with the polynucleotide andregenerating a transgenic plant from said plant cell; ii. crossing afirst plant comprising the polynucleotide with a second plant; or iii.genome editing a polynucleotide sequence encoding an Axmi205 toxin in atransgenic plant.
 15. The method according to claim 14, wherein themethod further comprises obtaining a transgenic progeny plant for one ormore generations from the transgenic plant, wherein the progeny plantcomprises the polynucleotide and has increased resistance to acoleopteran insect pest.
 16. A polynucleotide comprising a nucleotidesequence encoding the modified Axmi205 toxin according to claim
 4. 17. Anucleic acid molecule comprising the polynucleotide according to claim16 operably associated with a heterologous promoter.
 18. A transgenicplant comprising the nucleic acid molecule of claim
 17. 19. Thetransgenic plant according to claim 18, wherein the transgenic plant isa maize plant.
 20. A transgenic seed of the transgenic plant accordingto claim 19, wherein the seed comprises the nucleic acid molecule.